Reverse breeding

ABSTRACT

A method for efficiently producing homozygous organisms from a heterozygous non-human starting organism, comprising providing of a heterozygous starting organism; allowing the starting organism to produce haploid cells; creating homozygous organisms from the haploid cells thus obtained; and selecting the organisms having the desired set of chromosomes, wherein during production of the haploid cells no recombination occurs in order to obtain a limited number of genetically different haploid cells. Recombination can also be prevented or suppressed.

The present application is a continuation of U.S. patent applicationSer. No. 10/487,468 filed Apr. 3, 2006 now U.S. Pat. No. 8,242,327 whichis a national stage application of International Application No.PCT/EP02/09526, filed on Aug. 23, 2002, published as WO 03/017753 onMar. 6, 2003, and claiming priority to European Patent Application Nos.02075582.3 filed Feb. 12, 2002 and 01203193.6 filed Aug. 23, 2001.

FIELD OF THE INVENTION

This invention relates to a method for efficiently producing homozygousorganisms from a heterozygous non-human starting organism. The inventionrelates in particular to the use of this method in plant breeding toproduce parental lines for the production of hybrid offspring. Theinvention further relates to DNA constructs for use in this method, toprimer pairs to select genes for use in this method, to F1 hybridorganisms obtainable by crossing organisms that are the result of themethod and to seeds resulting from the method.

BACKGROUND OF THE INVENTION

Plant breeding is one of the oldest accomplishments of man. It beganwhen he domesticated plants were domesticated by growing them undercontrolled conditions and selecting those types that provided adependable source of food. The most important feature contributing tohigh yield of many new varieties is their hybrid nature. The mostdramatic example is hybrid corn, which was first introduced insignificant amounts in 1932 and now makes up about 95% of the acreage ofcorn in the United States. Hybrid varieties are now available in cropssuch as sorghum, sugar beet, sunflower, onions, castor beans, oilseedrape, leek, cucumber, tomato, spinach, melon, pepper, carrot, cabbage,cauliflower, broccoli, radish, egg plant etc., in fungi, such asmushrooms, and in animals, such as poultry and fish.

J. Sneep and A. Hendriksen (1979, Pudoc, Centre for AgriculturalPublishing and Documentation Wageningen), teach several methods forplant breeding which have been successfully applied during the lastdecades and which result in the varieties that are grown nowadays. Inthe Chapter “Current breeding methods”, J. Sneep and A. Hendriksen(1979) (supra, pp 104 233), describe general breeding techniques butalso the specific breeding technologies for a number of crops, such aspotato, sugar beet, maize, sunflower etc.

In general, selections are made from a collection of plants that can bederived from seeds from the market (commercial varieties), gene bankaccessions, land races etc. From this collection, the “best” plants areselected and crossed according to the art. So traditionally, pure linesor homogenous populations are obtained by breeding.

Plant breeding has the objective to produce improved crop varietiesbased on the exploitation of genetic variation, which exists within thegerm plasm of a plant species. Genetic variation is traditionallyobtained by crossing two genetically distinct plants to create hybridprogeny. The genotype of a progeny plant is the result of thecombination of the genotypes of the male and female gamete, whichthrough fusion resulted in a zygote, from which ultimately the progenyplant developed. Gametes are formed by the gametophytic generationduring the life cycle of a plant and therefore the genetic variation ofthe gametes is reflected in the genotypes of the gametophytes.Gametophytes differentiate from spores, which are produced by thesporophytic generation during the life cycle of the plant. Spores areproduced from differentiated cells in the reproductive organs of a plantthrough a specialized cell division process called meiosis.

During meiosis chromosomal segregation and recombination are theprocesses which cause independent re assortment and the generation ofnew combinations of the genetic factors of a diploid genome into ahaploid genome of the gametophytes. The genotype of one progeny plant isthe combination of genotypes of one male and one female gamete, whichfused to form a new sporophyte. Meiosis can therefore be considered tobe a pivotal process during the life cycle of any living organism tocreate genetic variability.

This variability is used to obtain desired plants with new properties.Often the combination of the different properties of the two parents ina hybrid is more advantageous than a homozygous (parental) plant. Theproduction of such hybrids is however rather complicated. In the case ofF1 hybrids, several putative parental lines are first made homozygous,e.g. by many generations of inbreeding and selection and subsequentlythey are crossed in various combinations to study their combiningability. The best combinations and their respective parental lines aresubsequently retained and give rise to a commercial F1 variety.

However, the normal way of obtaining desirable hybrids is rather timeconsuming since homozygous parental lines have to be produced first andthe desired combination of two of these homozygous parental lines hasthen to be selected. This process requires several generations.

Also in the case of animals, like for example farm animals, such ascattle, pigs, fish, such as salmon, and fungi, such as mushrooms,hybrids may be desirable but since animals take an even longer time tobecome sexually mature and reproduce it takes even longer to producehomozygous lines and select for the best combination of those to producea hybrid. Examples of animals for which the invention may be usefulinclude rainbow trout and aquarium fish such as zebrafish.

SUMMARY OF THE INVENTION

It is therefore a first object of the present invention to provide analternative method for providing homozygous parental lines for theproduction of hybrids.

It is a second object of the invention to use this method to provideeven more flexibility in combining desirable parental traits inheterozygous off spring.

In this application sometimes reference is made solely to plants.However, in such cases one could also read fungi or animals, exceptwhere it is clear from the context that only plants can be intended.

According to the invention it was surprisingly found that the reverse oftraditional breeding is possible, i.e. starting with the heterozygousplant to produce homozygous parental lines. The homozygous parentallines can reconstitute the original heterozygous plant or animal bycrossing, if desired even in a large quantity. An individualheterozygous plant can surprisingly be converted in a heterozygous(F1-hybrid) variety without the necessity of vegetative propagation butas the result of the cross of 2 homozygous lines derived from theoriginal selected plant.

The present invention thus relates to a method for efficiently producinghomozygous organisms from a heterozygous non-human starting organism,comprising:

a) providing a heterozygous starting organism;

b) allowing the starting organism to produce haploid cells;

c) creating homozygous organisms from the haploid cells thus obtained;and

d) selecting the organisms having the desired set of chromosomes;

characterized in that during production of the haploid cells essentiallyno recombination occurs in order to obtain a limited number ofgenetically different haploid cells.

In a preferred embodiment of the invention recombination is at leastpartially prevented or suppressed in contrast to situations in which thestarting organism is selected for its inability to have recombinationupon the formation of haploid cells.

The method can be used for plants, fungi and animals except humans.

By preventing or suppressing recombination the normal variation thatarises in every natural cross can be limited or even avoided. As aresult thereof, the number of haploid cells having different sets ofchromosomes is considerably reduced. Because of this, the cell ororganism regenerated therefrom with the desired set of chromosomes canbe quite easily identified.

When the chromosome set of such cell or organism regenerated therefromis doubled a homozygous cell or organism arises. Such organism can thenbe used in crosses with another homozygous organism produced in the sameway from the same donor organism to produce a hybrid organism.

The “desired set of chromosomes” can be one of a number of variants. Incase the original starting hybrid is to be produced the two homozygousorganisms produced according to the invention should together have theexact set of chromosomes of the starting organism. This is achieved whenboth parents have the same set of chromosomes as the gametes that formedthe hybrid. However, it is also possible that the new maternal line hasonly some of the chromosomes of the original maternal gamete and theothers of the original paternal gamete (“chromosome substitution”). Inthat case the other parent should again have the complement thereof ifthe production of the same hybrid is desired.

It is however also possible to combine the new line which has one ormore but not all of the chromosomes of the original parent with adifferent parent in plant breeding. The new homozygous lines as such canthus be a newly desired end product. This applies to lines having theoriginal parental chromosome composition as well as to lines having anew combination of chromosomes.

Recombination can be prevented or suppressed by various means, inparticular through dominant transgenic approaches, dominant negativemutation or treatment with a chemical.

In a first embodiment, the prevention or suppression of recombination isachieved by interfering with one or more target genes involved inrecombination. The target genes can be involved in double strand breaks,chromosome pairing, crossing-over and separation of sister chromatids.

Target genes (GenBank accession nos.) involved in the formation ofdouble strand breaks are SPO11 (J02987.1), MER1 (M31304.1), MER2(M38340.1), MRE2 (D11461.1), MEI4 (M84765.1), REC102 (M74045.1), REC104(Z15007.1), REC114 (Z14315.1), MEK1/MRE4 (X63112.1), RED1 (X16183.1),HOP1 (J04877.1), RAD50 (X14814.1), MRE11 (U60829.1), XRS2 (L22856.1),identified in yeast, or their functional homologues from other species.

Target genes (GenBank accession nos.) involved in chromosome pairingand/or strand exchange are RAD54/TID1 (M63232.1), DMC1 (M87549.1), MND1(protein accession NP_(—)011332.1), SAE2 (U49447.1), SAE3 (U82546.1),RED1 (X16183.1), HOP1 (J04877.1), HOP2 (AF078740.1), REC8 (AJ223299.1),MER1 (M31304.1), MRE2 (D11461.1), ZIP1 (L06487.1), ZIP2 (proteinaccession: NP_(—)011265.1), MEI5 (L03182.1), RAD51 (X64270.1), RAD52(M10249.1), RAD55 (U01144.1), RAD57 (M65061.1), RPA (M60262.1), SMC3(Y14278.1), SCC1 (Y14280.1), MSH2 (M84170.1), MSH3 (M96250.1), MSH6(AL031545), PMS1 (M29688.1), MER3 (P51979), DDC1 (protein accessionNP_(—)015130.1), MMS4 (U14000.1), identied in yeast, SOLODANCERS(AJ457977.1), KU70 (AF283759.1), KU80 (AF283758.1) identified inArabidopsis thaliana, HIM6 (AY095296.1), CDS1 (Y60A3A.12), CDS2(T08D2.7), identified in Caenorhabditis elegans, SCP3 (X75785.10),identified in Rattus norvegicus, MEI218 (U35631.2), identified inDrosophila melongaster, or their functional homologues from otherspecies.

After recombination complexes are formed (double holliday junctions)these are processed to either crossing-over events or non-crossing-overevents (called gene conversion). Most recombination complexes lead togene conversion, whereas only a few crossing-over events lead torecombination. Interfering in this last phase of meiosis to have moregene conversion leads to a lower recombination frequency, and can beachieved via target genes (GenBank accession nos.) selected from thegroup consisting of SGS1 (U22341.1), MSH4 (U13999.1), MSH5 (L42517.1),ZIP1 (L06487.1), ZIP2 (protein accession: NP_(—)011265.1), MLH1(U07187.1), MEC1 (U31109.1), MLH3 (protein accession NP_(—)015161.1)from yeast, or their functional homologues from other species.

In the present invention use can be made of the above genes originatingfrom the organism in which they were first identified or thecorresponding genes in other organisms, such as plants, that have thesame name and/or the same function (called herein “their functionalhomologues from other species”). Functional homologues of the abovegenes that are involved in meiotic recombination constitute potentialtargets for modification in plants or other species in which meioticrecombination is to be suppressed. The fact that the products theyencode perform the same or a similar biological function does notnecessarily mean that the genes have a significantly higher level ofidentity than genes which are not functional homologous.

According to the present invention a (candidate) target gene is definedas a gene residing within the genome of an organism which uponquantitative and/or qualitative modification of its expression resultsin a modified meiotic process within said organism which ischaracterized by the formation of functional, haploid spores thatcontain a full set of chromosomes but which have not been subjected tomeiotic recombination or which have been subjected to a reducedfrequency of meiotic recombination as compared to the situation in whichsaid gene is not modified.

Different genes and their functional homologues, which can but notnecessarily need to be homologous, qualify as (candidate) target genes.The only common denominator of target genes of the invention is the factthat upon their modification meiotic recombination is suppressed.

Once a target gene has been selected for modification this can beachieved in various manners.

In a first embodiment interfering with the target gene consists ofpreventing transcription thereof. This can be achieved by means of RNAoligonucleotides, DNA oligonucleotides or RNAi molecules directedagainst the target gene promoter.

Alternatively, transcription is prevented by means of the expression ofa negatively acting transcription factor acting on the target genepromoter. Such negatively acting transcription factor can be natural orartificial. Artificial negatively acting transcription factors can beemployed by the overexpression of an engineered polydactyl zinc-fingertranscription factor coupled to a general transcription repressor.

According to a further embodiment, the interfering with the target geneconsists of destabilizing the target gene mRNA, in particular by meansof nucleic acid molecules that are complementary to the target gene mRNAselected from the group consisting of antisense RNA, RNAi molecules,Virus Induced Gene Silencing (VIGS) molecules, co-suppressor molecules,RNA oligonucleotides or DNA oligonucleotides.

In another embodiment the interfering with the target gene consists ofinhibiting the target gene expression product. This can be achieved bymeans of the expression product(s) of one or more dominant negativenucleic acid constructs, overexpression of one or more suppressors whichinteract with the target gene product, or by means of one or morechemical compounds.

Furthermore, interfering with the target gene can consist of theintroduction of one or more mutations into the target gene leading toperturbation of its biological function. The one or more mutations canbe introduced randomly by means of one or more chemical compounds and/orphysical means and/or insertion of genetic elements. Suitable chemicalcompounds are ethyl methanesulfonate, nitrosomethylurea, hydroxylamine,proflavine, N-methyl-N-nitrosoguanidine, N-ethyl-N-nitrosourea,N-methyl-N-nitro-nitrosoguanidine, diethyl sulfate, ethylene imine,sodium azide, formaline, urethane, phenol and ethylene oxide. Physicalmeans that can be used comprise UV-irradiation, fast-neutron exposure,X-rays and gamma irradiation. The genetic element is a transposon,T-DNA, or retroviral element.

Mutations may also be introduced specifically by means of homologousrecombination or oligonucleotide-based mutation induction.

According to a further embodiment of the invention,

the prevention or suppression of recombination is achieved by a chemicalcompound preventing the spindle from being formed or by a chemicalcompound inducing aneuploidy.

After the starting plant has been treated such that recombination isprevented or suppressed before or while haploid cells are being formed,these cells are isolated and used for regeneration of a complete plant.Such plant is haploid and can become diploid either spontaneously orthrough other means, such as treatment with colchicine.

Haploid cells can be derived from germ line cells such as spore mothercells or somatic cells that have become haploid by means of a natural orinduced process.

Once the haploid plant is diploidized it is homozygous for allchromosomes and it can be used for various purposes.

It is possible to derive the chromosome composition of the originalparents (so-called “original parental line rescue”) of the hybrid thatis the subject of reverse breeding by molecular analysis of either theseed coat or the endosperm. The endosperm contains a double maternalgenetic dosage. A quantitative assay can be used to determine whichchromosomes are derived from the mother (twice the dosage) and whichfrom the father (once the dosage). The seed coat is maternal andrepresents the chromosome composition that originates from the mother.

The production of F1 hybrids can now be done in completely the reverseorder. Instead of selecting so-called original parental lines, andtesting suitable combinations, a heterozygous plant with an expectedsuitable combination of allelic forms of genes is selected, andcorresponding parental lines that could be used for the production of F1hybrid seeds of the same plant are derived from this plant. This processis herein called “reverse breeding”.

Importantly, reverse breeding technology allows a significantflexibility in the plant breeding process because in addition to theefficient reproduction of the exact starting genotype, for eachindividual chromosome a choice can be made to retrieve it either in ahomozygous maternal, homozygous paternal or heterozygous form as will beexplained hereinbelow.

Effectively, reverse breeding can be performed by preventing meioticrecombination in combination with efficiency enhancing methods forgenerating the parental lines, which in a preferred embodiment concernsthe production of doubled haploid plants and/or molecular genotypingtechnologies. Other methods are second generation restitution and selfpollination. In the latter case, plants in which recombination has beenprevented or suppressed are selfed to produce selfing seed. Moleculargenotyping techniques are then used to identify the homozygous plants inthe S1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a partial nucleotide sequence of BoDMC1 (SEQ ID NO: 1);

FIG. 2 is a partial nucleotide sequence of BcDMC1 (SEQ ID NO: 2);

FIG. 3 is a partial nucleotide sequence of LeDMC1 (SEQ ID NO: 3);

FIG. 4 is a partial nucleotide sequence of SmDMC1 (SEQ ID NO: 4);

FIG. 5 is a partial nucleotide sequence of NtDMC1 (SEQ ID NO: 5);

FIG. 6 is a partial nucleotide sequence of BoSPO11 (SEQ ID NO: 6);

FIG. 7 is a partial nucleotide sequence of BcSPO11 (SEQ ID NO: 7);

FIG. 8 is a partial nucleotide sequence of AtMSH5 (SEQ ID NO: 8);

FIG. 9 is a result of a BLAST X analysis of the AtMSH5 partialnucleotide sequence (query) (SEQ ID NO: 27) showing the level ofidentity of the translated AtMSH5 sequence with known MSH5 orthologues(Sbjct) from Saccharomyces cerevisiae (SEQ ID NO:28), Homo sapiens, (SEQID NO: 29), Mus musculus and (SEQ ID NO: 30) Caenorhabditis elegans (SEQID NO: 31);

FIG. 10 is a Map of pRZ51. RB=right border, LB=left border,spec=spectinomycin/streptomycin resistance, 35S pr=CaMV 35S promoter,Bc-DMC=BcDMC1, OCS-ter=octopine synthase promoter, Pnos=nopalinesynthase promoter, NPTII=neomycin phosphotransferase II, Tnos=nopalinesynthase polyadenylation signal;

FIG. 11 is a Map of pRZ52. RB=right border, LB=left border,spec=spectinomycin/streptomycin resistance, 35S pr=CaMV 35S promoter,Bc-SPO11=BeSP011, OCS-ter=octopine synthase promoter, Pnos=nopalinesynthase promoter, NPTII=neomycin phosphotransferase II, Tros=nopalinesynthase polyadenylation signal;

FIG. 12 is a Map of pRZ54. RB=right border, LB=left border,spec=spectinomycin/streptomycin resistance, 35S pr=CaMV 35S promoter,AtMsH5=AtMSH5, OCS-ter=octopine synthase promoter, Pnos=nopalinesynthase promoter, NPTII=neomycin phosphotransferase II, Tnos=nopalinesynthase polyadenylation signal;

FIG. 13 is a partial nucleotide sequence of BoMSH5 (SEQ ID NO: 17);

FIG. 14 is a partial nucleotide sequence of LeMSH5 (SEQ ID NO: 18);

FIG. 15 is a partial nucleotide sequence of SmMSH5 (SEQ ID NO: 19); and

FIG. 16 is a partial nucleotide sequence of NtMSH5 (SEQ ID NO: 20).

DETAILED DESCRIPTION OF THE INVENTION

The invention thus relates to the prevention or suppression ofrecombination in a process for the production of haploid cells and tothe production of homozygous lines from these cells.

Haploid cells can be the result of meiosis or are derived from somaticcells. In the latter case chemical compounds can be used to haploidizethe cells. Alternatively, reductional grouping is induced. Inreductional grouping the chromosomes are distributed over the daughtercells without the help of the spindle fibres and without DNAreplication. After cell division haploid cells are formed. Reductionalgrouping can be induced by treating (root) meristem or protoplasts witha chemical, such as caffeine or by neutralizing the genetic target ofthe chemical with a gene construct. Expression of the construct must beinducible because constitutive expression of the neutralizing constructwould be lethal.

According to the invention haploid cells are however preferably derivedfrom a meiotic process. The process of meiosis forms the pivotal eventin the life cycle of living organisms at which genetic variation iscreated. Moreover it marks the transition between the diploid,sporophytic and the haploid, gametophytic generation which alternateduring the life cycle of a plant. The specialized cell in the femalereproductive organ which enters meiosis, which is called megasporemother cell, is embedded in the differentiated ovule inside the ovary.During ovule formation, a number of mitotic events lead to thedifferentiation of a single megaspore mother cell per ovule out of a fewarchesporial cells which develop from hypodermal cells.

Within the male reproductive tissues (the anthers), a similar processleads to the formation of microspore mother cells although thearchesporial cells undergo several rounds of mitosis beforedifferentiating into microspore mother cells. As a consequence eachanther contains a large number of microspore mother cells.

A few mutants of maize and Arabidopsis have been identified which aredisturbed in the early functions of these differentiation processes. Themacl (multiple archesporial cells) mutant of maize is disturbed in agene that plays a role in the withdrawal of hypodermal cells from themitotic into the meiotic pathway (Sheridan, W. F. et al (1999) Genetics153, 933-941). The spl (sporocyteless) mutant in Arabidopsis isdisturbed in the differentiation of mega- and microspore mother cellsfrom archesporial cells (Yang, W-C, et al (1999) Genes Dev. 13,2108-2117).

The macro- and microspore mother cells, collectively called meiocytes,undergo meiosis, which results in the formation of four haploid sporesper meiocyte. Three of the four female spores or megaspores degeneratethrough callose deposition. The surviving megaspore differentiates after3 nuclear divisions and subsequent cellularisation into the femalegametophyte or embryosac.

The four male spores or microspores usually remain together and form aso-called tetrad structure. Upon differentiation of the malegametophytes from the microspores, the tetrad structure is dissolved andthe male gametophytes or pollen behave as loose entities.

Although there are significant differences in the cellular processesleading to the formation of female and male meiocytes as well as thedifferentiation of the macro- and microspores into an embryosac andpollen, respectively, the cytological events which occur during femaleand male meiosis are very similar suggesting the involvement of commongene products.

However, this does not necessarily mean that each of the events duringfemale and male meiosis is controlled by identical genetic loci. Forexample, in Arabidopsis the ASK1 gene is specifically involved in malemeiosis (Yang, M. et al (1999) Proc. Natl. Acad. Sci. USA 96,11416-11421) whereas SWI1 (Motamayor, J. C. (2000) Sex. Plant Reprod.12, 209-218), DYAD (Siddiqi, I. et al (2000) Development 127, 197-207)and ANTIKEVORKIAN (Yang, W-C. and Sundaresan (2000) Curr. Opin. PlantBiol. 3, 53-57) are specific for female meiosis.

During meiosis a number of cytological phases are distinguished and foreach phase a number of mutants has been described in plants.

During the initial phase called meiotic Prophase a number of stages arediscerned. During the initial stage called Leptotene, the individualchromosomes which have been replicated and which consist of two sisterchromatids start to condense and become shorter and thicker.Simultaneously, the nuclear envelope starts to disintegrate and thehomologous chromosomes start to associate. The next stage is calledZygotene in which the chromosomes are fully condensed and in which thehomologous chromosomes align and start to form the so-calledsynaptonemal complex (SC). The dif1/syn1 mutant of Arabidopsis isimpaired in the formation of the SC (Bhatt, A. M. et al (1999) Plant J.19, 463-472; Bai, X. et al (1999) Plant Cell 11, 417-430). The DIF1/SYN1gene products are homologous to the yeast cohesin REC8/RAD21 whichfunction in synapsis and recombination. At Pachytene the formation ofthe SC is completed for all chromosomes. At this stage meioticrecombination occurs which is initiated by the formation ofdouble-stranded breaks followed by chromatid exchange between homologouschromosomes. The physical links that are established between thenon-sister chromatids and which persist even in the absence of thesynaptonemal complex are called chiasmata. During Diplotene andDiakinesis the chromosomes fully condense, the nuclear envelope hasdisappeared and the spindle fibers have been formed. Subsequently duringMetaphase I, the pairs of homologous chromosomes are located in theequatorial plane of the cell. Then, during Anaphase I, the homologouschromosomes, each consisting of two sister chromatids which may haveundergone a number of recombination events and are held together by acentromere, move towards the opposite cellular poles. During TelophaseI, the polar movement is completed, the spindle disappears and the cellstarts to divide.

Subsequently, these cells enter Prophase II that is characterized by thealignment of the condensed chromosomes on the equatorial plane. Aspindle complex is being formed. During Metaphase II the chromosomes arefully aligned at the equatorial plane and the spindle complex iscompleted. During the next phase, called Anaphase II, the centromeresdivide and the sister chromatids move towards opposite poles. InTelophase II this movement process is completed, the spindle complexstarts disappearing and cell division initiates. Subsequently, thechromosomes resume their Interphase appearance characterized by uncoiledchromosomes located inside the nuclear envelope.

The end product of meiosis II is a set of four genetically distincthaploid cells, which can undergo mitosis to develop into gametophytes.The gametophytes produce the gametes, which upon fusion leads to theformation of a zygote, which develops, into an embryo that can grow outinto the next generation sporophyte.

The genetic variation, which occurs in the sporophyte, is determined bythe genotypes of the female and male gametes that fused upon theformation of the zygote. Therefore this genetic variation is createdduring the formation of the female and male spores during meiosis whichleads to genetic re-assortment of the original parental chromosomes aswell as chromosomal regions due to recombination events.

Meiosis and meiotic recombination are intricate processes which havebeen studied to different degrees, at different levels in differentorganisms. The molecular mechanism through which meiotic recombinationoccurs is not yet entirely clear. One model is the double strand break(DSB) repair model according to which meiotic recombination is initiatedby the formation of double strand breaks (DSBs) in one of the twointeracting non-sister chromatids. The formation of the DSBs isinitiated by a protein. This protein has been identified in yeast and iscalled therein SPO11 protein. Homologues of the SPO11 protein of yeastwere found in Schizosaccharomyces pombe named REC12, Arabidopsis,Drosophila, Caenorhabditis, mouse and man. Arabidopsis is the onlyeukaryote known so far to contain 3 paralogous SPO11 genes. Thehomology, which resides within the SPO11 proteins, is confined to fiveconserved motifs.

Next to the DSB formation, an exonuclease activity evoked by a proteincomplex in which MRE11, RAD50 and XRS2/NBS1 of yeast participate,resects the 5′-ends of the break in the 3′-direction, which results in 23′-OH single stranded tails. One of these tails invades the doublestranded DNA of the paired chromatid through base pairing with thecomplementary strand. Strand invasion involves RecA-like proteins, ofwhich DMC1 is specific for meiotic recombination. Through a DNA repairmechanism a bimolecular intermediate containing two Holliday junctionsis formed which involves the proteins MSH4, MSH5 and MLH1 in yeast. AHolliday junction resolution system, containing resolvases, can resultin gene conversion or crossover.

A large number of proteins has been identified which are involved inthis process which can either be specific for meiotic recombination orcan be involved in mitotic DSB and mismatch repair as well. Homologuesof many of these proteins are being identified in plant systems likeArabidopsis thaliana and the corresponding genes have been cloned. Theplant homologues of the SPO11 protien have been identified inArabidopsis thaliana and are named AtSPO11 and AtDMC1 (Couteau, F. et al(1999) Plant Cell 11, 1623-1634). They are involved in bivalentstabilization and chromosomal segregation.

According to the invention recombination in the starting organism is tobe prevented or suppressed. This prevention or suppression can beattained at various levels of the recombination event. No recombinationcan occur when no double strand breaks are produced, when thecrossing-over is impaired and when the chromosomes cannot pair. Variousgenes are involved in all these events. Impairing the function of one ormore of these genes leads to prevention (on/off) or suppression (lowerlevel) of recombination. For the purpose of this application, such genesare called “(candidate) target genes”.

Interfering with the function of these genes can be achieved through anumber of approaches which are either based on homology dependent genesilencing mechanisms such as co-suppression, antisense downregulation orRNA interference or which are based on expression of proteins whichinterfere with the functionality of the target protein. The lattermethod is for example downregulation through a dominant negativeapproach.

In case use is made of a homology dependent gene silencing approach, thegene construct which is used to achieve the silencing effect shouldcontain a DNA fragment which has a percentage of identity at thenucleotide level with a region of the target gene which is sufficient todownregulate this target gene to the extent that it results in theformation of viable, haploid spores that contain a full set ofchromosomes which have not been subjected to meiotic recombination orwhich have been subjected to a reduced frequency of meioticrecombination as compared to the situation in which the target genes arenot downregulated. This result can either be achieved after selecting arandom fragment of the gene or by selecting those segments of the geneas silencing fragment which encode the conserved domains of the encodedprotein.

In case there is not a sufficient percentage of identity between thesilencing DNA fragment and a specific region of the functional homologueof the gene which resides in the genome of a given crop species toachieve a sufficient level of downregulation, a fragment of thefunctional homologue of the gene itself can be used to achievedownregulation of this functional homologue within the crop species fromwhich it has been derived.

Functional homologues, which reside in other crop species, can bedown-regulated using the silencing DNA fragment if there is sufficienthomology.

In a preferred embodiment of the invention, modification of the targetgenes is achieved by genetic engineering of the crop species. The natureof the modification of the target gene can either be downregulation,which means that the expression of the target gene is reduced or ectopic(over)expression, which means that the expression of the target gene isincreased, and optionally taking place at a time different from thenatural expression. In the case of ectopic (over)expression the targetgene involved in recombination has a repressor function.

In order to downregulate a target gene, various methods can be used thatare based on homology with the target gene.

In a particular embodiment the downregulation of the target gene isachieved through a method referred to as antisense technology. In thismethod a gene is expressed in its reversed orientation with respect to atranscriptional promoter. This can be achieved by introduction of a geneconstruct into the genome of a plant in which the segment of a gene,which is normally expressed as RNA, is reversed in its orientationrelative to a transcriptional promoter. Usually such a construct isreferred to as antisense construct. Upon expression of the antisenseconstruct in a plant, the plant produces RNA molecules that aresynthesised using the coding strand of the gene construct as a templateand therefore are complementary to the coding strand. Usually this typeof RNA is referred to as antisense RNA. The result of the expression ofan antisense construct is that the gene or genes which reside in thesame plant and which upon expression leads to the synthesis of RNAcomplementary to the antisense RNA are effectively silenced.

In another embodiment the downregulation of the target gene is achievedthrough a method referred to as cosuppression technology. In this methoda gene is expressed in the sense orientation with respect to atranscriptional promoter. This can be achieved by introduction of a geneconstruct into the genome of a plant in which the segment of a genewhich is normally expressed as RNA has the same orientation relative toa transcriptional promoter as in a native gene. Usually such constructis referred to as cosuppression or sense cosuppression construct.

Upon expression of the cosuppression construct in a plant, the plantproduces RNA molecules that are synthesized using the non-coding strandof the gene construct as a template and therefore are complementary tothe non-coding strand. Usually this type of RNA is referred to ascosuppression RNA. The result of the expression of a cosuppressionconstruct is that the gene or genes which reside in the same plant andwhich upon expression leads to the synthesis of homologous RNA areeffectively silenced.

In yet another embodiment the downregulation of the target gene isachieved through a method referred to as RNA interference (RNAi). RNAiis a general term which refers to a phenomenon in which double strandedRNA (dsRNA) molecules very effectively mediate gene silencing of geneswith homology to the dsRNA. Silencing of an endogenous gene triggered bydsRNA is the result of post transcriptional gene silencing which is aphenomenon in which an RNA transcript is synthesized and rapidly andspecifically degraded. RNAi has initially been demonstrated to operatein Caenorhabditis elegans (Fire, A. et al (1998) Nature 391, 806-811).RNAi has also been demonstrated to be effective in other organismsincluding plants (Chuang, C-F. and Meyerowitz, E. M. (2000) Proc. Natl.Acad. Sci. USA 97, 4985-4990). Transgenes designed to express RNA whichis self-complementary and thereby is able to form duplexes or hairpinRNAs were shown to be highly effective in triggering virus resistanceand gene silencing (Smith, N. A. et al (2000) Nature 407, 319-320).

In yet another embodiment of the invention suppression of the targetgene may be achieved through specific transcriptional silencing of thetarget gene via the promoter. This may be achieved through expression ofan RNAi construct which results in the synthesis of double stranded RNAmolecules of which the nucleotide sequence is identical to a part of thepromoter region of the target gene. The promoter region of a gene islocated upstream (with respect to the trancriptional direction) of theposition at which transcription of the gene is initiated.

In yet another embodiment of the invention, suppression of the targetgene may be achieved through a methodology generally referred to asVirus Induced Gene Silencing or VIGS (Ratcliff et al (2001) Plant J. 25,237-245). In such an approach effective and specific gene silencing isachieved by infection of a plant with a plant virus carrying an insertwhich is homologous to the gene which needs to be silenced. Theadvantage of the VIGS systems is that there is no need to develop aplant transformation protocol for the plant species for which silencingof a target gene is pursued.

In all these embodiments, the silencing construct (antisense RNA,co-suppression, RNAi or hairpin construct or VIGS vector) preferablycontains a DNA fragment that is identical to the target sequence (geneor promoter) that needs to be silenced. However, the percentage ofidentity may also range between 50 and 100%, preferably between 60 and100%, more preferably between 70 and 100%, even more preferably between80 and 100%, most preferably between 90 and 100%.

The length of the DNA fragment in the silencing construct should be atleast 20 nucleotides but can also be longer to a maximum of thefull-length target sequence which needs to be silenced.

The transcriptional promoter which is used to synthesize the silencingmolecule can be a constitutive promoter or a promoter which isdevelopmentally regulated. The promoter may also be inducible forexample by a chemical compound.

Preferably, but not necessarily the expression of the silencingconstruct and the target gene that needs to be silenced coincides. Thisdoes not apply to the silencing via the promoter of a gene because thisapproach is directed to avoiding transcription so that no transcript isformed. The other techniques neutralize the transcription product afterit is produced.

In yet another embodiment of the invention suppression of the targetgene may be achieved through specific silencing of the target gene byintroduction of RNA oligonucleotides (Tijsterman et al (2002) Science295, 694-697). This may be achieved through chemical synthesis of RNAoligonucleotides of which the nucleotide sequence is identical to a partof the promoter region or transcribed region of a target gene andintroduction of the silencing oligonucleotides into the cell. Theadvantage of this specific embodiment of the invention is that there isno need to adopt a transgenic route in achieving reverse breeding for aspecific target crop.

Like in the other embodiments that use homology dependent gene silencingmechanisms, the RNA oligonucleotide which is used to silence a targetgene preferably has a nucleotide sequence which is identical to a partof the promoter or transcribed region of the target gene that needs tobe silenced. However the percentage of identity may also range between50 and 100%, preferably between 70 and 100%, even more preferablybetween 80 and 100%, most preferably between 90 and 100%. The singlestranded RNA oligonucleotide may be identical to either the sense orantisense strand of the DNA of the promoter or transcribed region of atarget gene. Alternatively, instead of using single stranded RNAoligonucleotides, single stranded DNA oligonucleotides may be used ofwhich the nucleotide sequence is designed as if it would be an RNAoligonucleotide. Moreover both double stranded RNA as well as DNAoligonucleotides may be used.

The oligonucleotides can be introduced into plants or plant cells bymethods well known to the person skilled in the art. These may includebut are not limited to polyethylene mediated uptake in protoplasts orparticle gun mediated uptake in plants or plant parts.

When suppression of target genes is pursued through a homology basedmethod like RNA interference, VIGS, oligonucleotides or others it ispreferred to carry out a search for sequences that are homologous to thetarget sequence and that reside within the genome of the species to besubjected to recombination suppression or prevention. “Homologous” isintended to mean here “having a level of identity with the nucleic acidfragment which is used to effectuate the homology based suppression ofthe target gene that leads to suppression of the sequences outside thetarget gene”. In case such sequences are found it may be desirable touse another fragment of the target gene for design of the silencingsequence in order to avoid interference with other parts of the genome.

In another embodiment suppression of the activity of the target gene maybe achieved through overexpression of a dominant negative construct, aprocess well known to the person skilled in the art. In such approach agene encoding a protein or modified protein is overexpressed in the cropspecies in which a target gene needs to be suppressed according to thepresent invention. The gene which encodes such a protein usually isreferred to a dominant negative gene as the effect of (over)expressionis inherited as a dominant genetic factor and it is causing a specificloss of function. The transcriptional promoter which is used tosynthesize the dominant negative construct can be a constitutivepromoter or a promoter which is developmentally regulated. The promoterof the dominant negative construct may also be inducible for example bya chemical compound.

The expression of the dominant negative construct and the target genethat needs to be suppressed should be spatially and temporarilyregulated such that the target gene is effectively suppressed.Preferably but not necessarily, the promoter of the dominant negativeconstruct and the target gene are regulated such that they are expressedin essentially the same part of the plant at essentially the same time.

According to yet another embodiment of the invention suppression oftarget genes is achieved through overexpression of a natural suppressorof the target gene. Such a suppressor may be a negatively actingtranscription factor that acts on the promoter of target genes or aprotein which interacts with the gene product of the target genes insuch a way that this gene product can not fulfil its natural function.The expression of a suppressor construct and the target gene that needsto be suppressed should be spatially and temporarily regulated such thatthe target gene is effectively suppressed. Preferably but notnecessarily, the promoter of the suppresser construct and the targetgene are regulated in an highly similar manner in terms of their spatialand temporal activity, i.e. they are expressed essentially at the sametime in the same part of the plant. The promoter of the suppressorconstruct may also be inducible for example by a chemical compound.

In a specific embodiment of the present invention, a silencing constructis used that modifies a target gene which specifically results insuppression of either female or male meiotic recombination. This can beachieved by interfering with the activity of a gene product that isspecifically active in either female or male meiotic recombination.Alternatively, this can be achieved by using a silencing construct thatis specifically active during female or male meiosis. The latter kind ofconstruct can either interfere with a target gene that is specificallyinvolved in female or male meiotic recombination or a target gene thatis involved in both female and male meiotic recombination.

This specific embodiment of the invention has practical utility when thesuppression of meiotic recombination leads to a reduction of the qualityof the spores, such as a reduced number of functional haploid spores.Plants that are suppressed in female meiotic recombination but not inmale meiotic recombination can be used as efficient pollinators toproduce new hybrids and vice versa, the plants that are suppressed inmale meiotic recombination can be used as efficient female lines duringthe production of new hybrids.

In case transgenic approaches are used for preventing or suppressingrecombination, so-called chimeric gene constructs can be made usingstandard molecular cloning techniques well known to the person skilledin the art and which can for example be found in Sambrook, J andRussell, D. W.: Molecular cloning, a laboratory manual (third edition),Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Suchconstructs are “chimeric” in the sense that they consist of various DNAfragments originating from various sources.

The chimeric constructs which are made to modify the activity, inparticular transcription or translation, but also transcript processing,protein modification, protein targeting, complex formation and activity,of the target genes usually comprise a promoter sequence and apolyadenylation signal sequence that are operably linked to the DNAfragment that is being used to achieve suppression of meioticrecombination such that a functional chimeric gene construct isproduced.

In order to transfer a chimeric gene construct into the genome of aplant, transformation vectors are prepared using standard molecularcloning techniques well known to the person skilled in the art and whichcan for example be found in Sambrook, J and Russell, D. W.: Molecularcloning, a laboratory manual (third edition), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

Promoter sequences which can be used according to the present inventioninclude but are not limited to constitutive promoters like the CaMV 35Spromoter (Odell, J. T. et al (1985), Nature 313, 810-812), theArabidopsis Actin 2 promoter (An, Y. Q. (1996) Plant J. 10, 107-121),the maize Ubiquitine 1 promoter (Drakakaki, G. et al (2000) TransgenicRes. 9, 445-452), the rice Actin 1 promoter (McElroy, D. et al (1990)Plant Cell 2, 163-171) and the Arabidopsis Farnesyl diphosphate synthase1S promoter (Cunillera, N. et al (2000) Plant Molec. Biol. 44, 474-485)or developmentally regulated promoters like the Arabidopsis Actin 11promoter (Huang, S. et al (1997) Plant Molec. Biol. 33, 125-139), theArabidopsis DMC1 promoter (Klimyuk, V. I. and Jones, J. D. (1997) PlantJ. 11, 1-14) or the Arabidopsis SPO11-1 promoter (Grelon, M. (2001) EMBOJ. 3, 589-600).

Other developmentally regulated promoters may be derived from the targetgene itself.

Inducible promoter systems which can be used are the ethanol induciblegene switch system (Caddick, M. X. et al (1998) Nat. Biotechnol. 16,177-180) and the glucocorticoid inducible system (Schena, M. et al(1991) Porc. Natl. Acad. Sci. USA 88, 10421-10425).

Polyadenylation sequences which may be used in constructs of the presentinvention include but are not limited to the Agrobacterium octopinesynthase polyadenylation signal (MacDonald et al (1991) Nucleic Acids.Res. 19, 5575-5581), the pea ribulose bisphophate carboxylasepolyadenylation signal (Hunt, A. G. and MacDonald M. H. (1989) PlantMolec. Biol. 13, 125-138).

According to a further aspect of the invention prevention or suppressionof recombination can be achieved by randomly inducing changes in thegenome of the organism and selecting those mutants that have acquired achange in the target gene that leads to the desired suppression orprevention of recombination.

In a preferred embodiment of this aspect of the invention, modificationof the target genes is achieved by mutagenesis of the crop species.Random mutations can be introduced into a plant genome by chemical meanslike treatment with ethyl methanesulfonate or nitrosomethylurea, byMorphogenics technology (BioWorld Today (2000), 11(108), 1-2) orphysical means like UV-irradiation, fast-neutron exposure or insertionalmutagenesis using transposons or T-DNAs. Specific mutations can beintroduced into a plant genome through homologous recombination(Paszkowski, J. et al (1988) EMBO J. 7, 4021-4026; Mengiste, T. andPaszkowski, J. (1999) Biol. Chem. 380, 749-758); Vergunst, A. C. andHooykaas, P. J. J. (1999) Crit. Rev. Plant Sci. 18, 1-31) oroligonucleotide-based mutation induction (Oh, T. J. and May, G. D.(2001) Curr. Opin. Biotechnol. 12, 169-172).

Plants in which the target gene is mutated can be readily identified byscreening methods like TILLING (Colbert, T. (2001) Plant Physiol. 126,480-484) or DELETAGENE (Li, X. et al (2001), 12th Internationalconference on Arabidopsis research (Abstract nr. 2)) which allow todetect aberrations residing within the target gene.

Preferably a mutant is selected in which the modification of the targetgene is conditional, which means that the mutant phenotype only becomesmanifest upon exposure of the plant to a specific environmentalcondition like a specific temperature. This allows to induce themodification only by exposing the plant to the specific environment.Under the conditions in which the modification is not manifest, themutant can be used for normal crossing and seed production.

In yet another preferred embodiment of the invention, modification ofthe target genes is achieved by treatment of the crop species withspecific chemical compounds which through interference with the productsof the target genes result in inhibition or reduction of meioticrecombination. An example of such chemical compound is etoposide whichthrough inhibition of the topoisomerase-II results in inhibition ofmeiotic recombination (Russell, L. B. et al (2000) Mutat. Res. 464,201-212).

In yet another preferred embodiment of the invention, aneuploidy can beinduced chemically by treatment of pre-meiotic cells with certainchemical compounds. This may be done by chemical treatment of floralbuds containing these pre-meiotic cells by submergence or spraying. Suchmethod may effectively be applied to modify meiotic recombination as hasbeen shown in patent application WO0054574. The mechanism by which achemical compounds induces aneuploidy is not always clear but there isexperimental evidence that aneuploidy can occur through interferencewith the spindle mechanism during mitosis and meiosis, fragmoplastfunction and chiasmata formation. The chemical that may be applied toinduce aneuploidy is selected from but not limited to chemicals such asetoposide, podophyllin, benomyl, maleic hydrazide, atrazine, butachlor,APM, griseofulvin, vinblastin-sulphate, diazepam, colchicine, cadmiumchloride, econazole, pyrimethamine, thiabendazole, thimerozal ornocodazole. Further details on aneuploidy inducing chemical compoundsand their mode of action as well their effective concentration can befound in C. B. S. R. Sharma (1990) Mutagenesis 5, 105-125 and referencestherein as well as in Sandhu et al (1991) Mutagenesis 6, 369-373.

After treatment of floral buds with the chemical compound which inducesaneuploidy, spores can be isolated from the treated buds which can beinduced to regenerate. Homozygous plants may be obtained throughdoubling of the chromosome number e.g. through treatment with colchicinein case spontaneous doubling has not been taken place already. Thepopulation of double haploid plants obtained through this method can beanalysed for the presence of a full complement of chromosomes bymolecular detection of marker alleles known to reside on a specificchromosome.

Gynogenesis is particularly suitable for applying reverse breedingeffectuated by chemical treatment. By using such method the specificchemical may be applied through the tissue culture medium used to applygynogenesis. It may also be possible to treat the sterilised ovariesdirectly with the chemical compound that prevents meiotic recombination.The reason for this particular form of gynogenesis to be suitable forreverse breeding is that meiosis is still taking place in some if notall ovules of the ovary tissues taken as explant for the gynogenesetissue culture.

Other culture techniques that allows in vitro manipulation prior to thestage in which meiotic recombination takes place are also suitable foruse in the invention.

In the above the haploids were the result of meiosis. However, it isalso possible to start with somatic cells for the production of haploidcells.

Accordingly, in yet another preferred embodiment of the invention, thegeneration of plants containing unrecombined original parentalchromosomes can be achieved by treatment of plants, plant organs orplant cells with chemical compounds like caffeine which result inchromosomal separation within a cell without the help of spindle fibers.In most cases the chromosomes separate evenly into two groups whichafter cytokinesis leads to haploid cells. The chromosomes in these cellscan be doubled e.g. by colchicine in case spontaneous doubling has notbeen taken place already and regenerated into plants.

As the chromosomes separate without recombination, their constitution isstill the same as in the original parent. Furthermore, as haploid cellsare formed, doubling of the chromosomal number leads to fully homozygousplants. However the distribution of the chromosomes is random andtherefore the resulting homozygous plants can contain all possiblecombinations of maternal and paternal chromosomal pairs.

This method is a specific embodiment of reverse breeding wherein somaticcells are used to produce progenitor cells which contain a haploidnumber of chromosomes which are unrecombined. This method is thus a formof reverse breeding in which there is no need to suppress meioticrecombination. This demonstrates that reverse breeding is a novelbreeding concept which can be effectuated by seemingly differentapproaches.

Once according to the present invention a silencing construct has beenprepared, which upon expression in a target crop species modifies theexpression of genes in a quantitative and/or qualitative way which canresult in the formation of viable, haploid spores that contain a fullset of chromosomes which have not been subjected to meioticrecombination or which have been subjected to a reduced frequency ofmeiotic recombination as compared to the situation in which these genesare not modified, such construct needs to be transformed into the cropspecies which is to be treated according to the present invention.

Currently many different technologies exist which allow the delivery,stable integration and expression of DNA molecules in the genome ofplants. These plant transformation technologies need to be combined withappropriate tissue culture technologies in order to regenerate a plantcell which has been transformed with a specific gene construct into atransgenic plant.

A well known plant transformation technology is based on the naturalability of a bacterial species called Agrobacterium tumefaciens todeliver and stably integrate a segment of DNA into the genome of a plantcell (Zambryski, P. et al (1989) Cell 56, 193-201). This piece of DNA,called T-DNA, is usually located on a plasmid which resides inside thebacterial cell. The natural T-DNA does not contain functions importantfor DNA delivery or integration and can in principle be any DNA. Theplasmid which contains the T-DNA can be a binary vector (Bevan, M.(1984) Nucleic Acids Res. 12, 8711-8721) or a cointegrate vector(Fraley, R. T. et al (1983) Proc. Natl. Acad. Sci. USA 80, 4803-4807).

Agrobacterium cells which contain a plant transformation vector can beco-cultivated with explants derived from leafs or seedlings in order todeliver the T-DNA in cells present in the explant. (Horsch, R. et al(1985) Science 227, 1229-1231).

Incubation of the explants on tissue culture medium results inregeneration of cells present in the explant through organogenesis orembryogenesis. In many systems this regenerative step is preceded by acallus phase which is variable in length. Usually the T-DNA contains aselectable marker gene which upon expression in the transformed plantcell can confer resistance to a phytotoxic compound like the antibioticskanamycin or hygromycin or the herbicides glyphosate orgluphosinate-ammonium. Addition of these phytotoxic compounds to thetissue culture medium during the regeneration of the cells of theexplants prevents the outgrowth of untransformed cells or transformedcells which do not express the selectable marker gene to an insufficientextent.

Following this principle many transformation protocols have beendeveloped for different crop species like potato (De Block, M. (1988)Theoretical and Applied Genetics 76, 767-774), lettuce (Michelmore, R.(1987) Plant Cell Reports 6, 439-442), tomato (McCormick, S. (1986)Plant Cell Reports 5, 81-84), pepper, cucumber (Trulson, A (1986)Theoretical and Applied Genetics 73, 11-15), carrot (Scott, R. J. andDraper, J. (1987) Plant Molecular Biology 8, 265-274), cauliflower (DeBlock, M. (1988) Plant Physiol. 91, 694-701), broccoli (Christy, M. C.and Earle, M. D. (1989) Australian Society of Plant Physiologists, 29thAnnual Meeting, Abstract 40), eggplant (Guri, A. and Sink, K. C. (1988)J. of Plant Physiol. 133, 52-55), sugar beet (Gasser, C. S. and Fraley,R. T. (1989) Science 244, 1293-1299), asparagus (Conner, A. J. et al(1988) Ninth Australian Plant Breeding Conference, Proceedings.Agricultural Research Institute, Wagga Wagga, pp. 131-132 sunflower(Bidney, D. (1992) Plant Mol. Biol. 18, 301-313), oilseed rape (ThomzikJ. E. (1995) Methods Mol. Biol. 44, 77-89, maize (Ishida, Y (1996) Nat.Biotechnol. 14, 745-750), wheat (Cheng, M. et al (1997) Plant Physiol.115, 971-980), rice (Chan, M. T. (1993), Plant Molec. Biol. 22,491-506).

Alternative methods to carry out plant transformation includestransformation of protoplasts in which DNA delivery is mediated bycalcium, polyethylene glycol, or electroporation (Pazkowski et al (1984)EMBO J. 3, 2717-2722; Potrykus et al (1985) Molec. Gen. Genet. 199,169-177; Fromm et al (1985) Proc. Natl. Acad. Sci. USA 82, 5824-5828;Shimamoto (1989), Nature 338, 274-276). Further methods includetransformation mediated by silicon carbide whisker (Dunwell, J. M.(1999) Methods Mol. Biol. 111, 375-382), microinjection (Holm, P. B. etal (2000) Transgenic Res. 9, 21-32) or biolistics (Klein et al (1988)Proc. Natl. Acad. Sci. USA 85, 4305-4309; Becker, D (1994) Plant J. 5,299-307). All these methods are useful in the present invention.

Transformants of crops that acquired a silencing construct are initiallyidentified by their phenotype of resistance to the selective agent whichhas been used to obtain selective regeneration of transgenic cellsexpressing the selectable marker gene. Subsequently, the resistanttransformants are further characterised molecularly to investigate thepattern of integration of the transformed DNA. Many techniques likepolymerase chain reaction (PCR) or Southern blotting are available tocarry out such an analysis and are well known to those skilled in theart (see for example techniques described Sambrook, J and Russell, D.W.: Molecular cloning, a laboratory manual (third edition, 2001), ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Thosetransformants that contain a single, intact copy, of the transformed DNAare preferably selected for further analysis. However, transformantscontaining multiple copies of the transformed DNA may be useful as well.In order to analyse whether the transformed DNA in the genome of atransformant is expressed, the transformant can be analysed on RNA orprotein species expected to be modified as a consequence of the presenceof the transformed DNA.

Techniques well known to those skilled in the art enable to analyse thetransgenic plant on the expression of the introduced gene or the effectthe expression of the introduced gene has on the expression of a targetgene by means of northern blotting, RT-PCR, in situ hybridisation, microarrays, western blotting, enzymatic activity assays.

Phenotypic changes which result as a consequence of the modification ofthe expression of a target gene may occur as well but this is notnecessarily the case. The fact that suppression of meiotic recombinationthrough downregulation of a target gene can result in phenotypic changesis illustrated by the example of a knock out mutation in the AtSPO11-1gene of Arabidopsis (Grelon, M. et al (2001) EMBO J. 20, 589-600). As aconsequence of the reduced or zero expression of the AtSPO11-1 gene inArabidopsis, the formation of bivalents at the end of the meioticProphase I is severely diminished. This can be explained by assumingthat the stabilisation of the bivalents is reduced as a consequence ofthe absence of meiotic crossing over events and thereby chiasmata.Despite this abnormality, the chromosomes of this Arabidopsis mutant dosegregate during meiosis, albeit in random direction which results inmany unbalanced non-functional gametes. Macroscopically this can beobserved by the fact that such mutant plants are semi-sterile i.e. astrong reduction of functional pollen en embryosacs are formed andthereby seed set is severely reduced. As this reduced fertilityphenotype can be easily observed at the whole plant level, thisphenomenon allows to identify plants in which a target gene has beenmodified either through genetic engineering, mutagenesis or chemicaltreatment. Although this phenotypic effect was found in this particularexample it needs not necessarily always to be occurring to the sameextent upon the modification of this or other target genes in othersystems. In case semi-sterility occurs as a consequence of modificationof target genes in the way as it has been described for AtSPO11-1 inArabidopsis, the number of functional gametes is relatively lower as afunction of the number of haploid chromosomes.

The % of functional gametes can be estimated by the formula (½)^(n)×100%in which n is the haploid chromosomal number. In case the limitation inseed set is determined by the female gametes, the % of seeds that areformed can be calculated by the same formula. In case crops like sweetpepper (Capsicum annuum L.) with 12 haploid chromosomes show the samephenotype upon downregulation of the functional homologue of AtSPO11-1,such plant produces only 1/4096×100%=0.024% viable seeds. Such lowamount of viable seeds jeopardizes the industrial applicability ofsuppression of meiotic recombination in plant breeding. This problem canbe alleviated by regenerating spores of the plants in which meioticrecombination is suppressed into doubled haploid plants.

Thus, according to another aspect of the invention DH production is usedto improve efficiency of the present method. The production of diploidplants out of haploid spores is a tissue culture technique which is usedwidely in plant breeding to accelerate the production of plant which arecompletely homozygous. Usually this technology is referred to as doubledhaploid or DH technology.

In a haploid or monoploid plant, only one genome is present once. Thismeans that all genes are present in a hemizyous state. In lower plantorganisms, haploidy may be the predominant state; such is the case inthe gametophyte of mosses. In crop plants however, haploidy is not thepredominant state, except for the inconspicuous and parasiticgametophytes, the pollen grain, the pollen tube, and the embryo sac.

Haploid plants are usually sterile because of univalent chromosomes.However, doubled haploids that are obtained either by spontaneousdoubling of the haploid chromosome content or achieved by other meanssuch as chromosome doubling agents are among the most valuable tools inplant breeding. Doubled haploid plants are genetically homozygous andtherefore the ultimate pure breeding lines that can theoretically onlybe achieved by many generations of inbreeding.

A haploid plant develops from haploid cells from an unfertilized ovule(gynogenesis), or haploid cells from anthers (androgenesis). Thefrequency of natural haploids is fairly low, about 1 per 1000 in thecase of parthenogenesis and about 0.1 per 1000 in the case ofandrogenesis. Because of the low efficiency of natural occurringhaploids, in vitro tissue culture methods have been worked out over theyears to provide plant breeders with sufficient numbers of doubledhaploids in order to partially or completely replace inbreeding. Antherand microspore cultures are well established techniques which are usedfor the production of homozygous lines in many crop species, such asmaize (Zea mays L.): Gaillard et al. Plant Cell Reports: 10: 55-58(1991), rice (Oryza sativa L.): Raina et al. Plant Cell Reports: 6:43-45, (1987), oilseed rape (Brassica napus): Keller W. and Armstrong K.Z. Pflanzenzuchting 80, 100-108 (1978), barley (Hordeum vulgare L.):Ziauddin et al. Plant Cell Reports 9: 69-72 (1990), egg-plant (Solanummelongena L.): Tuberosa R. et al. Genet. Agr. 41; 267-274 (1987),broccoli (Brassica oleracea var. Italica): Takahata Y. and Keller W.Plant Science, 74, 235-242 (1991), safflower (Carthamus tinctorius L.):Plant Cell Reports 10: 48-51 (1991), asparagus (Asparagus officinalis)Pelletier G. et al. C. R. Ac. Sci. Paris. Ser. D 274, 848-851 (1972).

Haploids and doubled haploids can also be derived from gametophyticcells of the ovary in barley (Hordeum vulgare L.)) (San Noeum L. (Ann.Amelior. Plantes 26, 751-754 (1976)). Doubled haploid production viaovary cells is suitable for crop species that are in many cases notamenable for anther or microspore culture. Examples are sunflower(Helianthus annuus L.) Gelebart P. and San L. Agronomie, 7, 81-86(1987), sugar beet (Beta vulgaris L.) Hosemans D and Bossoutrot D. Z.Pflanzenzucht 91: 74-77 (1983), melon (Cucumis melo L.) Cuny et al.Agronomie, 12, 623-630 (1992), watermelon (Citrullus lanatus (Thunb.))Sari N et al. Scientia Horticulturae 82, 265-277 (1999), cucumber(Cucumis sativus L.) Dirks R. U.S. Pat. No. 5,492,827 (1995).

Doubled haploid plants derived from normal diploid donor plants areusually self pollinated and the resulting progeny is geneticallyidentical and homogeneous, that is there should be no geneticsegregation of alleles anymore. The combination of doubled haploidtechniques with the suppression of crossing-over according to theinvention provides very powerful new possibilities for plant breeding.All plants derived from doubled haploid techniques applied to plants(with any degree of heterozygosity) where crossing-over (chromosomerecombination) is eliminated are fully homozygous. This means that apopulation of DHs derived from a plant in which recombination wassuppressed, provides homozygous DH-plants that when crossed with anotherDH-plant from the same population results in generation of an F1 hybridthat is genetically identical to the individual plant that was used togenerate the DH-population.

For example, cucumber has 7 chromosomes as a haploid set. In thetheoretical case where a donor plant is heterozygous for genes on allchromosomes, and no crossing-over takes place, there are 128 differentdoubled haploid genotypes that possibly can occur, two of them beingidentical to the original parental plants that constituted the donorplant, in case the donor plant was derived from a cross between twohomozygous original parental plants.

In comparison: after self-pollination of the same plant (heterozygous onall chromosomes and no crossing over taking place) there are 2187different (diploid) genotypes that possibly can occur and the frequencyof each of the original parental genotypes, in case the donor plant wasderived from a cross between two homozygous original parental plants, isonly 1 in 16384 (=(0.25)7) of diploid progeny plants.

To reach a sufficient probability of finding the genotype that one islooking for, the number of DH or inbred plants produced has to bemultiplied with a factor. A reasonable factor is 3-4, giving a 95-98%chance of finding the desired genotype. Even with such a multiplier, theamount of DHs that should be produced are still industrially applicable,whereas in traditional self-pollination, the number of descendants to beproduced amounts to very high quantities, that normally will not fitwithin the scope of a commercial breeding program.

Reconstruction of F1 hybrids by deducing the original parental ancestorsand creating new parental lines can be useful in case one would like todevelop alternative parents, that have better properties for seed orendosperm quality, for its commercial F1 seed production.

For reconstruction of an individual genotype, which is used fordownregulation of recombination with subsequent production of doubledhaploids, the genetic constitution is not relevant, and irrespective ofthe fact whether the plant is a hybrid or a plant with unknown geneticcomposition.

For instance in the case of cucumber there are theoretically 64different combinations of two doubled haploid (DH) lines that whencrossed provide progeny with a genotype identical to that of theoriginal plant. Thus, in a set of only 48 cucumber DH's with nearly 100%certainty a pair of DH's can be found that after crossing reconstructthe original donor genotype. Even for an economically important croplike corn with 10 chromosomes, only 98 doubled haploid combinations haveto be tested (see Example 2) to obtain parental lines that reconstructtheir progenitor, with a probability of 99%.

The exact retrieval of specific parental lines is desired in the specialcase of the said “transfer of cytoplasm” from one line to another whichis further described as example 12. The ultimate combination betweenrecombination suppression and the production of doubled haploidsfollowed by self pollination allows not only line selection but alsoprovides new lines that resemble the original parental lines in allpossible combination of chromosome combinations. As explained in theexample with cucumber (Example 2) it is possible to produce new parentalplants that when crossed reconstruct the donor material that was usedfor the derivation of the doubled haploids, whether or not the originalparents from the donor material were homozygous or heterozygous.

In addition to such lines, other lines are generated, that have 6chromosomes from one starting line and 1 chromosome from the otherstarting line that normally would generate the donor material (as ahaploid set). When the donor material used for recombination suppressionand doubled haploids, is an F1 hybrid that was created by crossing 2homozygous lines (for instance derived from doubled haploids) and thesaid invention that is disclosed here is applied, then parental linescan be recovered that are identical to the two starting lines.

In addition, combinations for every single chromosome pair from oneoriginal parent with the other set of other chromosome pairs from theother original parent are generated. Combinations as these can beobtained for every individual chromosome pair but also with doublepairs, triple pairs and so on can be obtained where the full set ofchromosomes is then eventually completed. In practice, this means thatparental lines are generated that are near to the original parentallines where only 1, or a limited number of the original parentalchromosome pairs is substituted by a chromosome pair from the otheroriginal parent. This allows the generation of many more combinations oforiginal parental lines than is possible in a traditional setting.Because of the absence of recombination and the fact that in diploidspecies the descendants of the DH technique are also fully homozygous,it is possible to detect genetic linkages.

In traditional genetics, the recombination frequency between 2 distinctgenetic loci is used as a measure of genetic distance between these locion a particular chromosome. The maximum frequency of recombinationbetween any two genes is 50%, the same value that would be observed ifthe genes were on non-homologous chromosomes and assorted independently.50% recombination occurs when the genes are so far apart on thechromosome that at least one crossing-over almost always occurs betweenthem. According to the invention, due to the lack of crossing-over,induced by mutation, chemical treatment(s) or transgenic means whetheror not stable or transient, all genes that reside on a particularchromosome are fixed in their respective allelic forms. Particularly incombination with doubled haploids, genes or loci located at the outerends of the chromosomes co-segregate. Co-segregation is easily monitoredif the genes encode visual markers, but currently available DNAfingerprinting technology association studies between DNA markers andimportant genes and DNA markers per se enhance the resolving power oflinkage analysis. Examples of such DNA fingerprinting technologies areRFLP (Restriction Fragment Length Polymorphism (Beckmann, J. S. andSoller, M. (1983) Theor. and Appl. Genet. 67, 35-43)), RAPD (RandomAmplified Polymorphic DNA (Welsh, J. and McClelland, M. (1990) NucleicAcids Res. 19, 861-866)), SSR (Simple Sequence Repeat (Wu, K-S. andTanksley, S. D. (1993) Mol. Gen. Genet. 241, 225-235)) and AFLP(Amplified Fragment Length Polymorphism, Vos, P et al (1995) NucleicAcids Res. 23, 4407-4414)).

It is obvious that reverse breeding technology has the potential ofcreating new varieties in time-frames that never have been possible andutilizing the maximum variation that occurs within an existing genepool.

According to a further aspect thereof, the invention relates to theimprovement of efficiency for transfer of cytoplasmic male sterility inplants by using suppression of “crossing-over” or recombination.Cytoplasmic male sterility or CMS is a trait that is widely used inplant breeding. CMS is used for making F1 hybrid varieties in vegetablespecies such as carrot, cabbage, cauliflower, broccoli, brusselssprouts, chicory and endive, but also in agronomic species such as sugarbeet and sunflower. CMS that is used in commercial plant breeding isinherited by the female parent, the phenotypic appearance of CMS (lackof pollen, brown anthers, petaloid anthers) can however also depend onnuclear factors that either may restore the male sterility or do notaffect sterility (so called maintainers of CMS) In order to add the CMStrait to a specific fertile breeding line, the person skilled in the artknows that several back crosses are required in order to replace themajority of the nuclear genome from one line that harbours CMS by thegenome that has to be converted to male sterility. The CMS donor line ismaintained by back-crossing with isogenic male fertile line(s).

The CMS donor is made homozygous for a recessive mutation or a transgenethat confers recombination suppression. The donor line is preferablygenetically dissimilar from the line that has to be converted to malesterility for a large number of nuclear genetic markers so that thedifference between the chromosomes of the CMS and the fertile donor canbe more easily determined. In order to convert a desired inbred line ora pure line (homozygous or nearly homozygous) into a similar line butwith a CMS background, a first cross is made by pollination of the saidCMS homozygous recombination suppressed line with pollen of the desiredline. The resulting F1 progeny contains CMS and 50% of the chromosomesof the desired line. In the meiosis of the resulting F1 plants, norecombination occurs as a result of the invention. This means that inthe egg cells, independent chromosome assortment takes place. In thecase of cabbage (Brassica oleracea L.) that has 9 chromosomes as ahaploid set this means that 1 in 512 egg cells ((½)⁹) have the same (buthaploid) genetic composition as the said desired line that was used forpollinating the recombination suppressed CMS line. This egg cell isagain fertilized by pollen of the desired line and the resulting seed isgenetically identical for the nuclear genes to the original desiredline, but now has acquired the CMS plasma. So in the second cross withthe desired line, 1 in 512 seeds is isogenic to the desired line butsaid having acquired the CMS plasma of the donor line. In the newlyachieved CMS/nuclear composition, no transgenic genes/plants have to beretained due to segregation of the transgenic locus that is responsiblefor suppression of meiotic recombination and because the transgenicplants are not retained.

Identification of the new CMS/desired nuclear line combination is veryeasy when DNA fingerprinting technology is used. In a preferredembodiment, genetic markers are used that have the capacity to identifyevery individual chromosome. In a preferred embodiment, one singlehomozygous recombination suppressed CMS-donor line can be used formaking several (independent flowers) crosses with numerous said desiredlines.

Surprisingly, with the present invention it is also possible to converta CMS line into a maintainer line for those plant species in whichrestorer genes reside within the germplasm like in Brassica sp., carrotand radish. In order to apply the current invention with this objective,a fertile plant containing nuclear restorer genes and normal, non-CMScytoplasm, is transformed with a construct that confers suppression ofmeiotic recombination. Transformants harbouring such constructpreferably in a homozygous form are used as pollinator in a cross with aplant from a CMS line for which a maintainer line needs to be produced.The resulting hybrid plants will be male fertile as a consequence of thepresence of the restorer genes and contain the construct in aheterozygous form. As the construct is genetically dominant, thechromosomes of the hybrid plants which are 50% derived from the originalCMS line and 50% of the fertile plant containing the restorer genes,will not recombine during meiosis. Subsequently, such hybrid plant willbe used as a pollinator in a cross with the original plant harbouringthe restorer genes and the normal, non-CMS cytoplasm. Using molecularmarkers which allow specific detection of the chromosomes originatingfrom the original CMS line, progeny plants are selected which contain afull complement of the chromosomes originating from the original CMSline. These plants are then used to produce doubled haploid plants whichare selected for a full complement of chromosomes from the original CMSline using the same molecular markers. The resulting plants can be usedas maintainer for the original CMS line.

Preferably, DNA fingerprinting is used to improve efficiency of thepresent invention. In a preferred embodiment of the present invention,DH technology is used in combination with suppression of recombinationto obtain, in an efficient way, completely homozygous, diploid plantswhich have a full complement of chromosomes comprising a randomcombination of the chromosomes of the plants from which the doublehaploid plants were derived.

Although this embodiment is the most efficient one, the plants which arecompletely homozygous and which have a full complement of chromosomescomprising a random combination of the chromosomes of the plants inwhich meiotic recombination was suppressed can be identified usingalternative approaches. These alternative approaches comprise DNAfingerprinting technologies, which enables the person skilled in the artto determine the level of polymorphisms that exists between genomes ofany origin or complexity in a random fashion. In order to select thehomozygous plants, seeds are produced by self-fertilization of a plantin which meiotic recombination is suppressed. The collection of theseselfing seeds is used to grow the first inbred generation (S1). Withinsuch S1, the total number of different genotypes that exists is0.5(2^(2n)−2^(n))+2^(n), where n is the haploid number of chromosomes.Within this population 2^(n) is the number of different but completelyhomozygous genotypes whereas all other plants are heterozygous for avariable number of chromosomes. In order to identify the homozygousplants, DNA that is extracted from these plants is analysed by a DNAfingerprinting technology. The relative level of polymorphism's that ismeasured for each plant of the S1 reflects the level of heterozygocity.This allows enriching the S1 population for plants with a relativelyhigh level of homozygocity.

In order to identify the plants which are fully homozygous, markeralleles which have a known position on the genetic map of a given cropspecies can be tested for polymorphisms within the plant in whichmeiotic recombination is suppressed. In principle, when recombination isfully suppressed, the identification of a single polymorphic markerallele per chromosome which can be measured in a co-dominant fashion issufficient to identify the homozygous plants in the S1. As the frequencyof homozygous plants in an S1 decreases when the haploid number ofchromosomes increases, this approach requires more input of resourcewhen the crop species contains a higher haploid number of chromosomes.When a crop species has a number of n haploid chromosomes, the frequencyof homozygous plants in an S1 is 2^(−n). Once these markers areavailable for each chromosome, it can be determined for each plant ofthe S1 whether these marker alleles are homo- or heterozygously present.

As during meiosis recombination is fully suppressed, homozygocity of asingle marker allele is diagnostic for all loci on the same chromosomein terms of their homozygocity. This analysis allows identifying thehomozygous plants in the S1 and further allows classifying thehomozygous lines in complementation groups. Two plants are consideredcomplementary when upon crossing of these plants, the genotype of theplant in which recombination was suppressed is fully recovered.

This analysis further allows producing F1 hybrids in which anypredetermined set of chromosomes is present homozygously whereas allothers are present heterozygously.

According to the invention endosperm or seed coat analysis from F1hybrids can be used to determine maternal genotype. As described above,the availability of an assay to determine the presence of a minimum ofone co-dominant marker allele per chromosome allows the determination ofthe zygocity of each chromosome of a plant of the S1 population producedon a plant in which meiotic recombination is suppressed. Within thegroup of homozygous plants those plants which have the same genotype asthe mother plant of the plant in which meiotic recombination issuppressed can be identified by analyzing the DNA of the seed coat ofthe seed from which the plant was grown in which meiotic recombinationis suppressed. The DNA in a seedcoat is of maternal origin andtherefore, the available assays for the marker allele can be used toanalyze the seedcoat DNA that reveals the identity of the maternalalleles. The data resulting from such analysis can be used to identifythe homozygous plants in the S1 which have a genotype identical to themother plant of the plant in which meiotic recombination is suppressed.The plants which have the same genotype as the father plant are thoseplants which are fully complementary to the mother plant.

As an alternative approach, the plants which have a genotype identicalto the mother and father plants can be identified by analyzing theendosperm of the seed from which the plant was grown in which meioticrecombination is suppressed. As in endosperm tissues, the maternalgenome is present in twofold over the paternal genome, a quantitativemeasurement of the presence of the marker alleles in a total nuclear DNAextract of the endosperm reveals the identity of the maternal andpaternal alleles. The data resulting from such analysis can be used toidentify the homozygous plants in the S1 which have a genotype identicalto the mother or father plant of the plant in which meioticrecombination is suppressed.

In some species, 2n gametes from 2n parents (=unreduced gametes) ariseby abnormalities during meiosis. In the special case of “second divisionrestitution” unreduced gametes arise by an incomplete second division.The result is a dyad where both 2n cells are separated by a reductioncell wall. Gametes that are produced in such a way are homozygous in thecase of the absence of crossing-over and recombination. In the presentinvention we show how to manipulate the phase of the meiosis in order toprevent that recombination takes place. Plants regenerated from 2ngametes produced by said second division restitution in the absence ofrecombination are the functional equivalent of doubled haploid plants.For SDR see fi. Hermsen J. In: The potential of meiotic polyploidizationin breeding allogamous crops. Iowa State J. Res, Vol 58, No 4, pp421-435 (1984). Mok D, and Peloquin S. Heredity 35, 295-302 (1975).

The invention is suitable for use in all non-human organisms, inparticular in plants, especially in agriculture (potatoes, vegetables)and horticulture (vegetables, fruit, flowers) but also in potted plants,flower bed plants, shrubs, trees and fungi (mushrooms). Crop plants thatmay be subjected to the method of the invention comprise maize, wheat,rice, sugar beet, oilseed rape, ryegrass, sunflower, soybean, tomato,cucumber, spinach, pepper, petunia, potato, tobacco, eggplant, melon,carrot, radish, lettuce, vegetable Brassica species (cabbage,cauliflower, broccoli, kohlrabi, Brussels sprouts), leak, bean, endive,chicory, onion, potato, strawberry, radish, fennel, table beet, celery.

In many commercial plant species, such as many ornamental and woodyplants, vegetative or clonal propagation is the exclusive or dominantway of commercial propagation. In breeding programs of these species,superior genotypes are identified in segregating populations, e.g. in anF2, and these are then maintained and multiplied by vegetativemultiplication techniques.

In many of these species the method of vegetative propagation of(heterozygous) plants has become dominant because production of hybridvarieties through seeds (as is done in many annual and biannual crops)first requires several generations of inbreeding of parental lines,which in many woody and tree species would take too much time for anycommercial program. By means of vegetative propagation superiorgenotypes are multiplied into a stock of genetically identical plants,and no time is “lost” to produce parental lines as is the case inseed-propagated hybrid crops.

However, there are also clear disadvantages of vegetative propagation.First, the logistics of producing plants through vegetative propagationis much more difficult than through seeds. Seeds can be stored easily,and often without problems for a long time. Seeds can be sown whenevercommercial amounts of planting stock is required.

In the case of vegetatively propagated material it is much moredifficult to respond to varying commercial needs for new planting stock.Vegetative production is labour and technology-intensive, and thusrelatively expensive. Diseases, especially viruses, are a constantthreat to vegetative multiplication. Many viruses are not transmitted byseeds, but are easily transmitted to clonal offspring obtained byvegetative reproduction techniques. For this reason some countries havestrict quarantine regulations governing the importation of vegetativelyproduced plants.

Not all genotypes perform equally well in vegetative propagation. Someare difficult to propagate in this way. F.i. rooting ability of treecuttings varies between species and clones.

Through reverse breeding according to the invention the genotype of theheterozygous clonally propagated plant can now be resynthesized andhybrid seeds with the said genotype provided.

In the context of the present invention the following definitions apply:

-   -   Starting organism: heterozygous organism that is used as a        starting material in the method of the invention. The starting        organism is not necessarily the direct result of a cross between        two parents, but if so these parents are called the “original        parents” and a line of such original parents is called an        “original parental line”.    -   (New) parent: a homozygous organism resulting from the method of        the invention that can be used in a cross with a complementary        (new) parent to reconstruct the original starting organism. A        line of each (new) parent is called a “(new) parental line”.    -   It should be noted that the use of the word “parent” or        “parental line” in passages that do not directly describe the        invention need not be references to a new parent or parental        line.    -   Genotype: The genetic constitution of an individual organism.    -   Target gene: a gene residing within the genome of an organism        which upon modification of its expression results in a meiotic        process within said organism which is characterised by the        formation of spores that contain a set of chromosomes which have        not been subjected to meiotic recombination or which have been        subjected to a reduced frequency of meiotic recombination as        compared to the situation in which expression of said gene is        not modified.    -   Functional homologues: Genes with the same or similar functions        which can reside within one organism or can reside within        organisms belonging to different biological species.    -   Suppression of meiotic recombination: An event which leads to        the reduction, preferably absence of exchange of chromosome        fragments between two paired chromosomes during meiosis.    -   The present invention is further elucidated in the Examples that        follow and that are for illustration purposes only and are in no        way intended to limit the invention.

EXAMPLES Example 1

The Effect of Using Regeneration of Doubled Haploid Plants inCombination with Recombination Suppression

For reverse breeding to be commercially feasible the efficiency ofidentifying fully homozygous plants which are present in the offspringof transformants in which meiotic recombination is suppressed isimportant. This example shows the effect, in terms of the degree ofincreased frequency of homozygous plants in the offspring population ofplants in which meiotic recombination is suppressed, of the use of DHtechnology in combination with suppression of recombination as analysedfor different crop species.

When recombination is suppressed, a fully heterozygous plant, whichcontains a haploid chromosome number of n, is able to produce a maximumnumber of 2^(n) genetically distinct gametes. When such a plant isself-fertilised, progeny plants have a maximum genetic variability of0.5(2^(2n)−2^(n))+2^(n) different genotypes. Within this population2^(n) genotypically different but completely homozygous diploid plantsexist whereas all other diploid plants are heterozygous for a variablenumber of chromosomes.

The application of DH technology in combination with suppression ofmeiotic recombination results exclusively in progeny plants, which arecompletely homozygous. As these plants are derived from microsporesthrough e.g. androgenesis or megaspores through e.g. gynogenesis, themaximum number of genetically distinct diploid plants is identical tothe maximum number of genetically distinct haploid gametes which can beproduced by a plant in which meiotic recombination is suppressed whichis 2^(n).

Table 1 shows the result of this analysis.

TABLE 1 The effect of DH technology on the efficiency of pure linerecovery in a fully heterozygous plant (i.e. a plant that isheterozygous on every one of its chromosomes) in which meioticrecombination is suppressed, as a function of the haploid chromosomalnumber Maximum # of Maximum # of Efficiency genetically geneticallyimprovement Haploid Maximum # of distinct distinct, fully expressedchromo- Example of genetically progeny plants homozygous progeny as a/bdue somal a plant distinct after self- plants after to DH number nspecies gametes fertilisation (a) DH production (b) technology 1 2 3 21.5 2 4 10 4 2.5 3 8 36 8 4.5 4 16 136 16 8.5 5 Arabidopsis 32 528 3216.5 6 spinach, 64 2080 64 32.5 corn salad 7 cucumber, 128 8256 128 64.5barley, scorzonera 8 alfalfa, 256 32896 256 128.5 onion 9 cauliflower,512 131328 256.5 lettuce, sugar beet, carrot, broccoli, cabbage, radish,endive 10 maize, 1024 524800 512.5 asparagus, sorghum, Chinese cabbage,cocoa 11 banana, 2048 2098176 1024.5 watermelon, celery, parsley,fennel, common bean 12 tomato, 4096 8390656 2048.5 pepper, melon,potato, tobacco, rice, egg plant 13 cotton 8192 33558528 4096.5 14 Durum16384 1.34E+08 8192.5 wheat, pea, lentil

This analysis shows that for most if not all crops the use of DHtechnology has a profound effect on the efficiency of the recovery ofhomozygous plant as compared with the offspring obtained through selffertilization of the transformants in which meiotic recombination issuppressed.

As inferred from this analysis, the efficiency improvement depends onthe haploid chromosomal number of a given plant species and ranges fromone to three orders of magnitude (i.e. 10× to 1000×). It is concludedthat the combined use of meiotic recombination suppression and DHtechnology significantly improves the commercial and practicalfeasibility of the method of the invention.

Example 2

Analysis of the Probability of Finding in a Number k of DH-plants from aStarting Plant in which Recombination was Fully Suppressed aComplementary Combination of DH Plants, that After Crossing canResynthesize the Genotype of the Starting Plant, as a Function of theChromosome Number n

The present invention teaches the combined use of meiotic recombinationsuppression in combination with a technology for efficiency improvementlike DH technology to enable the conversion of a heterozygous plant intoan F1 hybrid variety by means of crossing parental lines obtained by thepresent invention as such.

In this example the analysis is shown of the probability of finding atleast one complementary combination of two doubled haploid plants (acombination that after crossing can ‘resynthesize’ the starting plant),as a function of the haploid chromosomal number n of a given plantspecies and the number k of DH-plants produced from a heterozygousstarting plant in which meiotic recombination is fully suppressed.

When the haploid chromosomal number of a given crop species is expressedas n, the maximum number of genotypes which are obtained from a plant ofthat crop species in which meiotic recombination is fully suppressed andfrom which double haploid plants are produced is 2^(n). The probabilitythat one randomly chosen pair of double haploid plants from thispopulation, upon crossing, results in an F1 hybrid which has a genotypeidentical to genotype in which recombination has been suppressed(original genotype) is ½ (because 2^(n)/(2^(n))²).

In case a total number of k doubled haploid plants is produced, thereexists a number of 1/21·k·(k−1) combinations of 2 genetically distinctdoubled haploid plants which can be crossed. The probability for anyrandomly chosen combination of 2 DH's that they are complementary (canresynthesize the original genotype after crossing) is (½)^(n). Thus theprobability for any randomly chosen combination of 2 DH's that they arenot complementary is 1−(½)^(n)=2^(n)−1)/2^(n). In case of k doubledhaploids, ½·k·(k−1) combinations can be made and therefore theprobability that within this DH-population no complementary DH's can befound is ((2^(n)−1)/2^(n))^((1/2k(k−1)) and therefore the probability ofthat at least one complementary combination of two DH's can be found is1−((2^(n)−1)/2^(n))^((1/2k(k−1)).

Using this formula the number of doubled haploid plants can becalculated for each crop species which need to be pair wise crossed inorder to maximise the probability to find the original genotype. Theresult of this analysis is shown in Table 2.

TABLE 2 The probability of finding at least one combination of twocomplementary DH′s, using the ‘reverse breeding’ technology, as afunction of the haploid chromosome number n and the number of availablerandomly produced doubled haploid plants k n/k 2 4 8 16 24 32 48 64 128256 7 0.008 0.046 0.197 0.610 0.885 0.980 1.000 1.000 1.000 1.000 90.002 0.012 0.053 0.209 0.417 0.621 0.890 0.981 1.000 1.000 11 0.0000.003 0.014 0.057 0.126 0.215 0.424 0.626 0.981 1.000 12 0.000 0.0010.007 0.029 0.065 0.114 0.241 0.388 0.863 1.000

This analysis shows that the original genotype is resynthesized as an F1hybrid according to the present invention with high probability using 48doubled haploid plants for cucumber, 128 for cauliflower and 256 fortomato, melon and sweet pepper.

Example 3

Molecular Cloning and Characterization of the Target Genes DMC1, SPO11and MSH5 from Arabidopsis thaliana, Brassica oleraceae, Brassicacarinata, Lycopersicon esculentum, Solanum melongena and Nicotianatabacum

Total DNA is extracted from plant tissues using the Genelute PlantGenomic DNA Kit (Sigma-Aldrich, Zwijndrecht, the Netherlands). The PCRreaction was carried out using a total amount of 30 ng DNA after whichthe reaction products were analysed on a 1% agarose gel. Total RNA isextracted from plant tissues using the commercially available RNeasyPlant Mini Kit from Qiagen (Valencia, Calif., USA). The purified RNA issubsequently treated with 1 μl of 10 units/μl Rnase-free DNase (RocheDiagnostics, Mannheim, Germany) in order to remove any residual DNA. TheRT-PCR reaction is carried out using Superscript™ One-Step RT-PCR withPlatinum® Taq from Invitrogen (Breda, the Netherlands), after which thereaction products are analysed on a 1% agarose gel. PCR products arecloned using the TOPO TA Cloning® system of Invitrogen (pCR®2.1-TOPO®)which is based on TA cloning and blue white colony screening.

1. Cloning of DMC 1

Based on the published gene sequence of DMC1 of Arabidopsis thaliana,AtDMC1 (GenBank Accession No. U76670), a primer combination wasdeveloped consisting of the following nucleotide sequences: forwardprimer 5′-ACAGAGGCTTTTGGGGAATT-3′ (SEQ ID NO:9) and reverse complementprimer 5′-ACAGAGGCTTTTGGGGAATT-3′ (SEQ ID NO:10). PCR analysis revealeda 380 bp cDNA fragment by RT-PCR from flower buds of Arabidopsisthaliana and an 1100 bp fragment from genomic DNA of Arabidopsisthaliana. This result is expected based on the known genomic sequence ofthe AtDMC1 gene. Sequence analysis of the cloned PCR products confirmedthe identity of the cloned fragment as being part of the AtDMC1 gene asthe obtained nucleotide sequence was identical to the publishedsequence. This result shows that the developed primer combination can beused effectively to specifically amplify a region of AtDMC1.

The same primer combination was used in an RT-PCR amplification reactionusing RNA extracted from flower buds of Brassica oleraceae and Brassicacarinata. For both plant species a 380 bp cDNA fragment was obtainedwhich was cloned and sequenced. The Brassica oleraceae DMC1 gene isdenominated BoDMC1 and its nucleotide sequence of the 380 bp cDNAfragment is shown in FIG. 1. The Brassica carinata DMC1 gene isdenominated BcDMC1 and its nucleotide sequence of the 380 bp cDNAfragment is shown in FIG. 2.

Sequence alignment of the obtained sequences with the AtDMC1 gene showeda very high degree of identity of BoDMC1, BcDMC1 and AtDMC1. Thepercentages of identity between the different sequences are as follows:AtDMC1 and BoDMC1 95%, AtDMC1 and BcDMC1 93%, BcDMC1 and BoDMC1 96%.

The same primer combination was used in a PCR amplification reactionusing genomic DNA extracted from tissues of Lycopersicon esculentum,Solanum melongena and Nicotiana tabacum which resulted in specificamplification products of 1100 bp for all 3 plant species. Thesefragments have a length which corresponds well to the length of thegenomic fragment of Arabidopsis thaliana and were denominated LeDMC1 forLycopersicon esculentum, SmDMC1 for Solanum melongena and NtDMC1 forNicotiana tabacum. The fragments were cloned and sequenced, the resultof which is shown in FIG. 3 for LeDMC1, FIG. 4 for SmDMC 1 and FIG. 5for NtDMC 1. A BLAST analysis showed that the fragments contain regionswith a high level of identity to AtDMC1 cDNA.

Together these data show that the cloned fragments of the solaneceousspecies are amplicons of the AtDMC1 orthologues which reside within thegenome of these species.

2. Cloning of SPO11

In order to isolate DNA fragments of orthologous genes of SPO11, aprimer combination was developed of which the primers correspond to aposition of the Arabidopsis thaliana SPO11-1 (AtSPO11-1, ACCESSIONAF-302928) genomic DNA which encodes a stretch of amino acids which ishighly conserved between known SPO11 orthologues of different species.The primers have the following nucleotide sequences: forward primer5′-AACGGGTTGGTGATGGG-3′ (SEQ ID NO:11) and reverse complement primer5′-CCATATGGATCACAGTCAAC-3′ (SEQ ID NO:12). PCR analysis revealed a 350bp cDNA fragment by RT-PCR from flower buds of Arabidopsis thaliana.This result is expected based on the known cDNA sequence of theAtSPO11-1 gene. Sequence analysis of the cloned PCR product confirmedthe identity of the cloned DNA fragment being derived from the AtSPO11-1gene, as the obtained nucleotide sequence was identical to the publishedsequence of AtSPO11-1. This result shows that the developed primercombination can be used effectively to specifically amplify a region ofAtSPO11-1.

The same primer combination was used in an RT-PCR amplification reactionusing RNA extracted from flower buds of Brassica oleraceae and Brassicacarinata. For both plant species a 350 bp cDNA fragment was obtainedwhich was cloned and sequenced. Sequence alignment of the obtainedsequences with the AtSPO11-1 gene showed a very high degree of identityfor both fragments with the AtSPO11-1 gene. The Brassica oleraceae SPO11gene is denominated BoSPO11 of which the nucleotide sequence of the 350bp cDNA fragment is shown in FIG. 6. The Brassica carinata SPO11 gene isdenominated BcSPO11 of which the nucleotide sequence of the 350 bp cDNAfragment is shown in FIG. 7.

The percentages of identity between the PCR-fragments are as follows:AtSPO11-1 and BoSPO11 94%, AtSPO11-1 and BcSPO11 93%, BoSPO11 andBcSPO11 99%.

3. Cloning of MSH5

In order to isolate part of the Arabidopsis thaliana MSH5 gene use wasmade of the algorithm Codehop (Rose et al (1998) Nucleic Acids Research26, 1628-1635). Based on conserved blocks of amino acids generatedthrough alignment of MSH5 orthologues of Caenorhabditis elegans, Musmusculus and Saccharomyces cerevisiae, a primer combination is generatedconsisting of a specific clamp and degenerate core region. The followingprimer combination was used to amplify a region of the Arabidopsisthaliana genome: forward primer5′-GTTTTTTATGGCTCATATTGGATGTTTYGTNCCNGC-3′ (SEQ ID NO:13) and reversecomplement primer 5′-TCCACAGTATTAGTTCCCTTTCCAWAYTCRTCDAT-3′ (SEQ IDNO:14), where Y stands for C or T, N stands for A, T, G or C, W standsfor A or T, R stands for A or G and D stands for A, G or T. PCRamplification using this primer combination of Arabidopsis thalianagenomic DNA resulted in a fragment of 220 bp which was cloned andsequenced. This sequence is given in FIG. 8.

A BLAST-X analysis revealed a high level of identity at the amino acidlevel of the translation product of the cloned fragment with known MSH5amino acid sequences which is shown in FIG. 9. This demonstrates thatthis method can be used effectively to specifically isolate a portion ofthe MSH5 orthologue of Arabidopsis thaliana which was named AtMSH5.

Based on the nucleotide sequence of AtMSH5, a specific primercombination was made to amplify additional plant MSH5 sequences. Thisprimer combination has the following sequence: forward primer5′-TgTCCCGGCTGCATCGGCCAAAATCGGC-3′ (SEQ ID NO:15) and reverse complementprimer 5′-GAATTCGTCAATCAAAATCAGTGACCG-3′ (SEQ ID NO:16) and generates afragment of 170 bp on Arabidopsis thaliana genomic DNA.

This primer combination was then used in a PCR reaction using genomicDNA of Brassica oleraceae, Lycoperisicon esculentum, Solanum melongenaand Nicotiana tabacum as template. For all plant species an amplifiedfragment of 170 bp was obtained.

These fragments were sequenced and the sequences were analyzed byBLAST-X. The result showed that the obtained fragments represent theMSH5 genes of the respective crop species. The genes were denominated asfollows: Lycoperisicon esculentum MSH5: LeMSH5 (FIG. 14); Solanummelongena MSH5: SmMSH5 (FIG. 15); Nicotiana tabacum MSH5: NtMSH5 (FIG.16) and Brassica oleracea MSH5: BoMSH5 (FIG. 13).

Example 4

Construction of RNA Interference (RNAi) Vectors for DownregulatingTarget Genes DMC1, SPO11 and MSH5

In order to downregulate the activity of a target gene in a particularplant species, use is made of RNA interference. For that purpose DNAfragments of the DMC1 and SPO11 of Brassica carinata and the MSH5 geneof Arabidopsis thaliana are inserted into pKANNIBAL (Wesley et al (2001)The Plant Journal 27, 581-590) such that upon expression in plants anRNA molecule is formed which folds back upon itself thus forming ahairpin structure that triggers the specific degradation of homologousRNA. The vector pKANNIBAL contains an intron positioned downstream ofthe CaMV 35S promoter and upstream form an octopine synthasepolyadenylation signal. At either side of the intron a multiple cloningsite is positioned which allows convenient insertion of the left andright arm of DNA corresponding to the RNA interference target in ainverted orientation relative to each other. Upon transcription theintron is removed by splicing and the left and right arm fold back oneach other forming the double stranded RNA.

In order to generate a left arm for DMC1, SPO11 and MSH5, the genefragments are reamplified from the vectors in which they have beencloned using primers which are extended with recognition sites for XhoIhybridising at the 5′-end of the gene fragment and KpnI hybridising atthe 3′-end of the gene fragment.

The fragments which are generated by PCR using these primers is digestedwith XhoI and KpnI and subsequently inserted in pKANNIBAL digested withXhoI and KpnI. The resulting plasmids are denominated pRZ039 containingDMC1, pRZ040 containing SPO11 and pRZ041 containing MSH5.

Subsequently, the right arms are prepared similarly but using adifferent set of primers which generate a XbaI site at the 5′ end of thegene fragment and a HindIII site at the 3′-end of the gene fragment.Upon digestion of the right arms they are inserted into the vectorscontaining the corresponding left arm resulting in pRZ042 for DMC1,pRZ043 for SPO11 and pRZ044 for MSH5.

As a final step the complete hairpin cassettes, containing the DMC1,SPO11 and MSH5 sequences as inverted repeat, are inserted separately asa NotI fragment into the NotI site of a T-DNA of a binary vector calledpART27 which contains the neomycin phosphotransferase II gene asselectable marker for plant transformation. The integrity of the T-DNAwas confirmed by sequence analysis. The resulting binary vectors,denominated pRZ051 for DMC 1 (FIG. 10), pRZ052 for SPO11 (FIG. 11) andpRZ054 for MSH5 (FIG. 12) are transferred into Agrobacterium tumefaciensusing a triparental mating procedure with the helper plasmid pRK2013(Ditta et al (1980) Proc. Natl. Acad. Sci. USA 77, 7347-7351).

Because of the high level of sequence identity of the BcDMC1 and BcSPO11and the AtMSH5 sequence with the respective orthologous genes, theconstructs are effective in the downregulation of the target geneswithin all species of the Cruciferaceae family. Moreover, as the LeDMC1,SmDMC1 and the NtDMC1 sequences show regions of high similarity to theBcDMC1 cDNA, pRZ051 are also effective in solaneceous species. Inaddition, given the similarity of the BcDMC1 to the DMC1 gene of ricethe BcDMC1 sequences can be used even more broadly i.e. also inmonocotyledonous plant species like for instance rice, wheat, barley andmaize.

In general, the above described method can be used to make constructscontaining DNA fragments which are homologous to other target genes thatneed to be down-regulated.

Example 5

Transformation of Arabidopsis thaliana with pRZ051, pRZ052 and pRZ054

Agrobacterium tumefaciens strain C58 (ATTC 33970) containing either oneof the plant transformation vectors pRZ051, pRZ052 or pRZ054 is grownovernight in LB medium containing streptomycin (100 mg/L) andspectinomycin (300 mg/L) to select for the vectors and rifampicin (40mg/L) and gentamycin (25 mg/L) to select for the Agrobacteriumtumefaciens C58 background at 29° C.

In order to produce transgenic Arabidopsis plants, the floral dip methodis used, as described by Desfeux et al. (2000) Plant Physiology 123,895-904. The bacterial cells are resuspended in floral dip solution (50g sucrose+500 μl Silwett L-77 surfactant (Helena Chemical Comp. Fresno,Calif., USA) per liter MilliQ™ (Millipore, Etten-Leur, the Netherlands).Bolting plants, containing multiple floral buds, are submerged into thedipping solution containing the Agrobacterium cells at an OpticalDensity (OD) between 1.0 and 1.5 during 5-10 seconds with gentleagitation.

After inoculation, the plants are contained in a plastic container tokeep high humidity under low light conditions for a day andsubsequently, seeds are grown on the plants.

Transformants are selected by germinating surface sterilised seeds in0.1% agarose layered upon half-strength MS plates containing 50 mg/Lkanamycin. Kanamycin resistant seedlings are transferred to soil in agreenhouse.

In total 51 kanamycin resistant seedlings/construct were grown to matureplants which were analysed by PCR for the presence of the T-DNA. Primercombinations were designed which specifically amplify either the NPTIIgene (NEO-FORW+NEO-REV), the region from the CaMV 35S promoter to theintron (35S-F1+RNAi-intr-R1) and the region from the intron to the OCSterminator (RNAi-intr-F1+OCS-R1). The sequences of these primercombinations are given below. The result of this analysis showed that inall plants specific amplification signals were obtained for thementioned primer combinations which confirms the transgenic status ofthe kanamycin resistant seedlings and which shows the presence of theRNA interference constructs. Sterile plants have been confirmed fromthis experiment.

NPTII: (SEQ ID NO: 21) NEO-FORW 5′-CAG ACA ATC GGC TGC TCT GAT GCC-3′(SEQ ID NO: 22) NEO-REV 5′-CGT CAA GAA GGC GAT AGA AGG CG-3′Promotor-Intron: (SEQ ID NO: 23) 35S-F1 5′-AgAATgCTgACCCACAgATggTTA-3′(SEQ ID NO: 24) RNAi-intr-R1 5′-CTTCgTCTTACACATCACTgTCAT-3′Intron-Terminator (SEQ ID NO: 25)RNAi-intr-F1 5′-ATgACAgTgATgTgTAAgACgAAg-3′ (SEQ ID NO: 26)OCS-R1 5′-TggCgCTCTATCATAgATgTCgCT-3′

Example 6

Transformation of Crop Plants and Production of Homozygous Lines

1. Constructs

The constructs described in Example 4 were used for the transformationof various crop plants by means of Agrobacterium. Arabidopsis constructscan be used in Brassica. Optionally, the genes of the constructs ofExample 4 can be exchanged with the homologous endogenous gene of therelevant crop as given in the description. In addition, functionalhomologues can be used.

2. Transformation and DH Production

2.1. Maize

Incorporation of silencing constructs in the genome of maize areperformed according to EP-801134, U.S. Pat. No. 5,489,520 or EP97114654.3 which teaches Agrobacterium transformation of DSM6009 cornprotoplasts. The silencing construct introduced into the maize cellsconfers an inhibitory effect when the regenerated transformed plantundergoes meiosis on recombination so that recombination is omitted orsignificantly reduced. As a consequence of the activity of the saidinhibitory nucleic acids, numerous egg cells respectively pollen, werefound to contain a chromosome number that deviates from the normalnumber and are partially or completely inadequate for either beingfertilised (egg cells) or as a functional pollinator (pollen). In thatcase the transformants are either male or female sterile or the seedproduction is lowered.

Some microspores respectively egg cells did however contain a normal,functional haploid set of chromosomes that results from a meiosis whereno or little recombination has taken place (as compared to wild type).These haploid microspores respectively egg cells are the startingmaterial for making doubled haploids.

Haploids in maize are obtained from microspores as described byPescitelli S and Petolino J (1988) Plant Cell Reports 7: 441-444;Coumans M et al., (1989) Plant Cell Reports 7: 618-621; Pescitelli S etal., (1989) Plant Cell Reports 7: 673-676. Buter B (1997) In: In VitroHaploid Production in Higher plants, vol 4, 37-71. Kluwer AcademicPublishers. Eds. S Jain, S Sopory & R Veilleux.

Subsequently diploid plants are produced from haploid plants by eitherspontaneous diploidization or chemically. Preferably, plants areselected that contain a single copy of the transgene. Due to reductionor elimination of recombination during meiosis some of these plants arehomozygous for all alleles. On average, 50% of those doubled haploidscontain the transgene that confers the recombination downregulationwhereas 50% is free of transgenic nucleic acids.

Alternatively, haploid maize plants were produced following natural andartificial pollination with a haploid inducer as described by RotarencoV (2002) Maize Genetics Cooperation News Letter 76: 16. In this caseseeds were obtained that contain haploid embryos. Also in this case,only haploids that have lost the transgene due to segregation from thehemizygous donor material are retained.

Chromosome doubling is performed as described by Wan, Y & Widholm, J(1995) Z. Pflanzenzuecht 114: 253-255.

Plants that contain one copy of the transgene (established by means ofSouthern blot or so-called Invader technology) are withheld for use incrosses in order to avoid repetitive transformation events.

2.2. Rice

Rice genetic transformation is carried out according to Zhang Bing andWei Zhiming (1999) Acta Phytophysiologica Sinica vol 25, no 4, or Dattaand Datta (1999) In: Methods in molecular biology vol 111, 335-347 Eds.Robert D. Hall, Humana press Totowa, N. J.

After the said inhibitory DNA that confers inhibition of recombinationduring meiosis is incorporated in the rice genome, preferentiallyregenerants containing one copy of the inhibitory DNA are further usedfor making doubled haploids by means of anther culture, microsporeculture and ovary culture according to Gosal S et al., (1997) In: InVitro Haploid Production in Higher plants, vol 4, 1-35. Kluwer AcademicPublishers. Eds. S Jain, S Sopory & R Veilleux.

2.3. Onion

The method of the invention is especially powerful in crops with arelatively low chromosome number. Onion (2n=2x=16) is therefore anexcellent species for practical application of the present invention.Transformation in onion is performed according to protocols developed byEady (1995) New Zealand Jounal of Crop and Horticultural Science, vol23: 239-250.

Again plants containing one copy of the silencing DNA constructconferring inhibition of recombination during meiosis are retained andused as starting material for making doubled haploids according toKeller E and Korzun L. (1996) In: In Vitro Haploid Production in Higherplants, vol 3, 51-75. Kluwer Academic Publishers. Eds. S Jain, S Sopory& R Veilleux.

Subsequently diploid plants are produced from haploid plants by eitherspontaneous diploidization or chemically.

2.4. Cucumber

Cucumber with a haploid chromosome number of 7 is also a crop specieswhere the invention is very powerful. The silencing constructs areintroduced by means of Agrobacterium transformation in embryogeniccallus as disclosed in EP-97114654.3 or by Agrobacterium transformationvia direct organogenesis according to Ganapathi A and Perl-Treves R. In:ISHS Acta Horticulturae 510: VII Eucarpia Meeting on Cucurbit Geneticsand Breeding; Mohiuddini A et al., (2000) Plant Tissue Cult 10 (2):167-173.

After identification of transformants with only one copy of thetransformed DNA that confers inhibition of recombination during meiosis,haploids are produced by means of gynogenesis as described in EP 0 374755.

Subsequently diploid plants are produced from haploid plants by eitherspontaneous diploidization or chemically.

2.5. Sugar Beet

Transformation in sugar beet is performed as described by Hall R et al.,(1996) Nature Biotechnology 14, 1133-1138.

Subsequently, doubled haploids are obtained as described in Pedersen Hand Keimer B (1996) In: In Vitro Haploid Production in Higher plants,vol 3, 17-36. Kluwer Academic Publishers. Eds. S Jain, S Sopory & RVeilleux.

2.6. Brassica sp.

Transformation of various Brassica species is performed according toMoloney M et al., ((1989) Plant Cell Reports 8, 238-242) for Brassicanapus; Metz T et al., ((1995) Plant Cell Reports 15, 287-292) forbroccoli (Brassica oleracea var. italica) and cabbage (B. oleracea var.Capitata); and Bhalla P and Smith N ((1998) Molecular Breeding 4,531-541) for cauliflower (Brassica oleracea var. Botrytis).

Doubled haploids were prepared according to Palmer C et al., (1996) In:In Vitro Haploid Production in Higher plants, vol 2, 143-172. KluwerAcademic Publishers. Eds. S Jain, S Sopory & R Veilleux.

2.7. Eggplant

Transformation of Solanum melongena is performed according to Leone etal. (1993) In: Biotechnology in Agriculture and Forestry, vol. 22, PlantProtoplasts and Genetic Engineering III, Y. P. S. Bajaj ed.,Springer-Verlag (Heidelberg), pp. 320-328. Doubled haploids are preparedaccording to Dumas de Vaulx, R. and Chambonnet (1982) Agronomie 2:983-988. Subsequently, diploid plants are produced from haploid plantsby either spontaneousdiploidization or chemically.

Example 7

Reverse Breeding for Transfer of CMS (Cytoplasmic Male Sterility)

CMS is one of the most prominent tools for plant breeding in theproduction of F1 hybrid varieties. Farmers demand a uniform phenotype(and therefore preferentially genotype) of the plant from the seeds theybuy. In order to achieve this, self pollination of the seed producingplant has to be excluded. In order to do this emasculation of the femaleline by hand is required which is a costly and error prone activity.

In some crops natural male sterility offers a better and more efficientalternative. Such crops are for instance but not limited to rice, sugarbeet, carrot, and Brassica spp. Until 1970 nearly all of the hybrid cornwas produced using T cytoplasm for F1 production.

A selected pure line as a result of traditional plant breeding orobtained by the aid of doubled haploid methodology that has beenpropagated by self-fertilization is converted to male sterility bymaking a cross of this line to a line that is a carrier of cytoplamaticsterility.

Preferably, the pollinator and the CMS donor are characterizedgenetically by using genetic markers such as but not limited to AFLP,RFLP, RAPD, Invader etc as is well known to the persons skilled in theart.

In this Example the male sterile line is suppressed for recombination,as exemplified in the other examples. When this suppression is achievedtransgenically then lines are selected that are homozygous for thetransgene. The F1 progeny that results from the cross of the pollinatorwith the CMS acceptor inherits 50% of the chromosomes form both parents.Egg cells produced by plants of this generation are formed in theabsence of recombination which means that when the haploid chromosomenumber is 9 which is the case for cauliflower, carrot and sugar beet, 1egg cell in 512 of the egg cells that contain a full chromosome setinherits exactly the same chromosomal constitution as the egg cells orthe pollen from the pollinator. This means that after successfulpollination already the second back cross gives rise to seeds in whichthe chromosomal content of the original pollinator has been transferredin the cytoplasmic environment of the CMS line.

The identification of this isogenic line is performed with the aid ofmolecular makers.

Example 8

Using the Invention to Produce a Maintainer Line (B-line) from aHomozygous CMS-line (A-line)

A maintainer or B-line of Daucus carota, Brassica oleracea or Raphanussativus was produced starting from a homozygous cytoplasmic male sterileline or A-line using the present invention. In many crops cytoplasmicmale sterile (CMS) mother lines are used to produce hybrid seeds. TheCMS mother line (A-line) is maintained by backcrossing with a line thathas the same or highly similar nuclear constitution, but has normalplasma, and thus is male fertile (B-line). Often a new A-line isproduced by crossing elite male fertile genotypes with CMS-plants,choosing those combinations that maintain the CMS in the offspring(B-lines, i.e. lines with ‘maintainer-capacity’, i.e. lines that lackrestorer genes), and backcrossing the CMS progeny several times with theoriginal B-line until the resulting A-line is genetically very similarto the B-line. Surprisingly in species in which restorer genes arepresent (Brassica, carrot, radish) reverse breeding can provide acorresponding B-line from any homozygous CMS plant by means of thebreeding scheme given below. The symbols use in the breeding scheme areas follows:

Example 9

Reverse Breeding Using Caffeine Treatment of Meristematic Cells

Seeds of Brassica oleraceae are surface sterilised by submerging themfor 30 minutes in 6% solution of hypochlorite (commercial bleach, 1.5%NaOC1 final concentration) after which they are thoroughly rinsed withsterile milliQ. Subsequently, the seeds are germinated on sterile, wetfilter paper. The germinated seeds which show the primary root aresoaked into a 70 mmol/L caffeine solution for a period of 2 hours afterwhich the seeds were rinsed with sterile milliQ.

Subsequently the seeds are allowed to recover by placing them onsterile, wet filter paper for 24 hours. The optimal treatment fordifferent plant species can differ and should be established by testingdifferent concentrations of caffeine, different incubation times anddifferent recovery times. After the treatment, the meristematic cellsare taken into tissue culture by preparing the root tips andtransferring them onto MS-medium containing 0.5 μg/l 2,4-D (2,4dichlorophenoxy acetic acid) into the dark for a period of 2 weeks toinduce callus.

After this callus induction, plants are regenerated by placing thecallus onto medium containing 0.5 mg/L BA (16/8 hours light/dark, 25°C.). After regeneration the shoots are analysed molecularly for thepresence of each of the haploid chromosomes by using genetic markers foreach chromosome, preferably markers that are polymorph for each set ofchromosomes. Haploid shoots containing a full complement of chromosomesare doubled by treatment with colchicine.

Example 10

Reverse Breeding by Chemical Induction of Aneuploidy Followed bySelection of Haploid Plants Containing a Complete Set of Chromosomes

Flowering plants of Brassica oleraceae which contain young floral budswhich are in a pre-meiotic state are treated with different chemicalcompounds known to induce aneuploidy selected from etoposide,podophyllin, benomyl, maleic hydrazide, atrazine, butachlor, APM,griseofulvin, vinblastin-sulphate, diazepam, colchicine,cadmiumchloride, econazole, pyrimethamine, thiabendazole, thimerozal ornocodazole according to C. B. S. R. Sharma (1990) Mutagenesis 5, 105-125and references therein; and Sandhu et al. (1991) Mutagenesis 6, 369-373.

The chemical is applied by dipping the pre-meiotic floral buds into asolution or by spraying a solution onto the pre-meiotic floral buds. Asthe developmental stage of the floral buds of a plant may be variableand therefore the effectiveness of the applied chemical may be differentfor each individual floral buds, the treatment is repeated a number oftimes to enhance the probability of exposing the appropriatedevelopmental stage for a maximal number of floral buds. In addition tothe chemical compound, the solution contains a surfactant like Agralin(Syngenta, Roosendaal, the Netherlands) (0.25 ml/100 ml).

After application, the treated buds are labelled and grown to the stageoptimal for microspore regeneration which on average occurs when thebuds have a length of approximately 3 millimeter. Purified microsporesare harvested from these buds and given a stress treatment of 2 days at32° C. which is optimal to induce sporophytic development of the haploidcells.

After regeneration the shoots are analysed for the presence of each ofthe haploid chromosomes as described above. Haploid shoots containing afull complement of chromosomes are doubled by treatment with colchicine.

Example 11

Using the Invention to Provide Seed Propagated Varieties in Species thatNow are Commercially Multiplied by Vegetative Propagation Techniques

In many commercial plant species, e.g. many ornamental and woody plants,vegetative or clonal propagation is the exclusive or dominant way ofcommercial propagation. In breeding programs of these species, superiorgenotypes are identified in segregating populations, e.g. in an F2, andthese are then maintained and multiplied by vegetative multiplicationtechniques, which are well known to the person skilled in the art. Inmany of these species the method of vegetative propagation of(heterozygous) plants has become dominant because production of hybridvarieties through seeds (as is done in many annual and biannual crops)first requires several generations of inbreeding of parental lines,which in many woody and tree species would take too much time for anycommercial program. By means of vegetative propagation superiorgenotypes are multiplied into a stock of genetically identical plants,and no time is “lost” to produce parental lines as is the case inseed-propagated hybrid crops.

However, there are also clear disadvantages of vegetative propagation.The logistics of producing plants through vegetative propagation is muchmore difficult than through seeds. Seeds can be stored easily, and oftenwithout problems for a long time. Seeds can be sown whenever commercialamounts of planting stock is required. In the case of vegetativelypropagated material it is much more difficult to respond to varyingcommercial needs for new planting stock. Furthermore, vegetativeproduction is labour- and technology-intensive, and thus relativelyexpensive. Diseases, especially viruses, are a constant threat tovegetative multiplication. Many viruses are not transmitted by seeds,but are easily transmitted to clonal offspring obtained by vegetativereproduction techniques. For this reason some countries have strictquarantine regulations governing the importation of vegetativelyproduced plants. In addition, not all genotypes perform equally well invegetative propagation. Some are difficult to propagate in this way.E.g. rooting ability of tree cuttings varies between species and clones.Through reverse breeding according to the invention the genotype of theheterozygous clonally propagated plant can now be resynthesized andhybrid seeds with the said genotype provided.

Transformation of Malus domestica is performed according to Yepes, L. M.and H. S. Aldwinckle. 1989. Genetic transformation of apple. Abstract.UCLA Symposium on Plant Gene Transfer. Park City, Utah, Apr. 1-7, 1989.

Doubled haploids are prepared according to Zhang et al. (1992) PlantBreeding 108:173-176. Subsequently, diploid plants are produced fromhaploid plants by either spontaneous diploidization or chemically.

Example 12 Using Reverse Breeding to Improve Seed Production in HybridCrops

Production of hybrid seeds at a commercial scale can encounter a largenumber of difficulties which leads to a reduced quality or quantity ofseeds. This can hinder in turn the commercialisation of high qualityhybrids varieties. These difficulties can be caused by a number ofdifferent factors like an intrinsic poor seed production capacity of thematernal line of the hybrid or a difference in flowering time, cropheight or flower morphology (preventing insects of carrying out crosspollination because of a preference for one flower type over the other)of the maternal and paternal lines of the hybrid.

By applying reverse breeding according to the invention to a hybridwhich has excellent agronomic properties but poor seed productioncharacteristics (which makes the commercialisation of the hybrid lessattractive or even impossible), this hybrid can be resynthesized usinglines which differ from the original maternal and paternal lines. Byselecting a combination of lines which allows the production ofcommercial seeds of both high quality and quantity, thecommercialisation of the hybrid becomes economically feasible or moreattractive.

The invention claimed is:
 1. A method for producing homozygous plantsfrom a heterozygous starting plant, comprising: at least partiallypreventing or suppressing recombination by interfering with at least onetarget gene involved in double strand breaks, chromosome pairing and/orstrand exchange while allowing the heterozygous starting plant toproduce haploid cells, whereby a limited number of genetically differenthaploid cells are thus obtained; creating homozygous plants from thehaploid cells by forming doubled haploid cells; and selecting from theplants those having the desired set of chromosomes, wherein theinterfering with the at least one target gene includes destabilizing theat least one target gene mRNA or transcript.
 2. The method of claim 1wherein the at least one target gene mRNA or transcript is destabilizedby nucleic acid molecule(s) complementary to the at least one targetgene mRNA or transcript selected from the group consisting of antisenseRNA, an RNAi molecule, an RNA oligonucleotide, and a Virus Induced GeneSilencing (VIGS) molecule.
 3. The method of claim 2 wherein the nucleicacid molecule(s) complementary to the at least one target gene mRNA ortranscript comprises a VIGS molecule.
 4. The method of any one of claims1-3, wherein the at least one target gene is selected from the groupconsisting of SPO11, RHD54/TID1, DMC1, SAE3, RED1, HOP1, HOP2, REC8,MER1, MRE2, ZIP1, ZIP2, MEI5, RAD51, RAD52, RAD54, RAD55, RAD57, RPA,SMC3, SCC1, MSH2, MSH3, MSH6, PMS1, SOLODANCERS, HIM6, CHK2, MER2, MEI4,REC102, REC104, REC114, MEK1/MRE4, RAD50, MRE11, XRS2, and theirfunctional homologues.
 5. The method of any one of claims 1-3, furthercomprising crossing the homozygous plants to produce a progeny plant. 6.The method of any one of claims 1-3, further including transferringcytoplasmic male sterility (CMS).
 7. The method of any one of claims1-3, including making parental lines to produce F1 hybrid seeds.